Transparent exopolymer particle production and aggregation by a marine planktonic diatom (Thalassiosira weissflogii) at different growth rates

Transparent exopolymer particles (TEP) play an important role in the ocean carbon cycle as they are sticky and affect particle aggregation and the biological carbon pump. We investigated the effect of growth rate on TEP production in nitrogen limited semi‐continuous cultures of the diatom Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle. Steady‐state diatom concentrations and other indicators of biomass (chl a, and total carbohydrate) were inversely related to growth rate, while individual cell volume increased with growth rate. There was no change in total TEP area with growth rate; however, individual TEP were larger at high growth rates and the number of individual TEP particles was lower. TEP concentration per cell was higher at higher growth rates. SYTOX Green staining showed that <5% of the diatom population had permeable cell membranes, with the proportion increasing at low growth rates. However, TEP production rates were greater at high growth rates, refuting our hypothesis that TEP formation is dependent on dying cells with compromised cell membranes in a diatom population. Measurements of particle size distribution in the cultures using laser scattering showed that they were most aggregated at high growth rates. These results indicate a coupling between TEP production and growth rate in diatoms under N limitation, with fast growing T. weissflogii producing more TEP and aggregates.     

STUDIES ON TRANSPARENT EXOPOLYMER PARTICLES (TEP) PRODUCED IN THE ROSS SEA (ANTARCTICA) AND BY PHAEOCYSTIS ANTARCTICA (PRYMNESIOPHYCEAE)1

The distribution and production of transparent exopolymer particles (TEPs) were studied quantitatively both in cultures of Phaeocystis antarctica Karsten (Prymnesiophyceae) and in natural phytoplankton assemblages in the Ross Sea, Antarctica. TEP production in culture was a function of growth rate and photosynthetic activity and was strongly influenced by photon flux density. The concentrations of TEP measured during a bloom, dominated by P. antarctica, were higher than those produced by coastal diatom blooms and were correlated with chlorophyll a (Chl a), being low at Chl a levels below 3 μgL^?1 but increasing rapidly at greater Chl a concentrations. Because higher chlorophyll hek are dominated 4 larger P. antarctica colonies, this relationship suggests that TEP was produced primarily by sloughing and disintegration of the colonial matrix. TEP concentrations (both absolute and relative to Chl a) increased as the bloom's biomass increased. Vertical distributions of TEP and Chl a showed TEP: chlorophyll maxima at the bottom of the water column at most stations. Because TEP and floc formation are tightly coupled, we suggest that mucous flocs derived from TEP, rather than intact P. antarctica colonies, are the dominant component of aggregates and subsequent organic carbon vertical flux.     

Inhibition of copepod feeding by exudates and transparent exopolymer particles (TEP) derived from a Phaeocystis globosa dominated phytoplankton community

We investigated if (1) dissolved compounds excreted by Phaeocystis globosa and (2) transparent exopolymer particles (TEP) formed from carbohydrates excreted into the water affect the feeding of nauplii and females of the calanoid copepod Temora longicornis during a P. globosa bloom. Copepod grazing on the diatom Thalassiosira weissflogii in the presence of these possible grazing deterrents was measured during three successive weeks of a mesocosm study, simulating the development of a P. globosa bloom. Our results demonstrate no indication for the presence of feeding deterrents in the dissolved phase, but a strong inhibitory effect of transparent exopolymer particles (TEP) on the consumption of algae by both nauplii and adult copepods. The inhibitory effect of TEP was connected to the accumulation of DOM during the progress of the bloom. We suggest that a reduction in the grazing pressure of zooplankton may increase the survival of the liberated single cells during disruption of colonies and allow seeding populations to persist. Furthermore, P. globosa reduces the trophic efficiency of the food web not only by withdrawal of its colonies from grazing but also by a relaxation of the grazing pressure on co-occurring phytoplankton and by alteration of the food web structure via TEP production.     

Seasonal microbial community dynamics correlate with phytoplankton-derived polysaccharides in surface coastal waters

Phytoplankton produce large amounts of polysaccharide gel material known as transparent exopolymer particles (TEP). We investigated the potential links between phytoplankton-derived TEP and microbial community structure in the sea surface microlayer and underlying water at the English Channel time-series station L4 during a spring diatom bloom, and in two adjacent estuaries. Major changes in bacterioneuston and bacterioplankton community structure occurred after the peak of the spring bloom at L4, and coincided with the significant decline of microlayer and water column TEP. Increased abundance of Flavobacteriales and Rhodobacterales in bacterioneuston and bacterioplankton communities at L4 was significantly related to the TEP decline, indicating that both taxa could be responsible. The results suggest that TEP is an important factor in determining microbial diversity in coastal waters, and that TEP utilisation could be a niche occupied by Flavobacteriales and Rhodobacterales.     

Community composition and activity of prokaryotes associated to detrital particles in two contrasting lake ecosystems

The composition, distribution and extracellular enzyme activities of bacteria attached to small (2-50 microm in size) transparent exopolymer and Coomassie-stained proteinaceous particles (TEP and CSP) were examined in two lakes of different trophic status located in the Massif Central of France. TEP concentrations (10(4)-10(6) particle per L) were significantly higher in the more productive lake and were significantly related to chlorophyll a concentrations. The majority of TEP and CSP were colonized by bacteria that constituted 2.6% and 7.4% of the total 4',6-diamidino-2-phenylindole-stained bacteria in lakes Pavin and Aydat, respectively. In both lakes, the composition of particle-associated bacteria was different from that of free-living bacteria, the Betaproteobacteria and Bacteroidetes (i.e. former Cytophaga-Flavobacteria group) being the dominant groups on particles. We also found that 2-5 microm TEP were more colonized than 2-5 microm CSP in the two lakes, and that TEP colonization was higher in the less productive lake. Measurements of Leucine aminopeptidase and alpha-glucosidase activities in fractionated lake water (0.2-1.2, 1.2-5 and >5 microm fractions) indicated that proteolytic activity was always higher and that particle-associated bacteria have higher enzymatic activities than free-living bacteria. The glycolytic activities in the 1.2-5 and >5 microm fractions were related to the abundance of TEP. We conclude that small freshwater detrital organic particles constitute microhabitats with high bacterial activities in pelagic environments and, undoubtedly, present significant ecological implications for the prokaryotic community structure and function in aquatic ecosystems.     

Production of transparent exopolymer particles (TEP) by benthic suspension feeders in coastal systems

In the marine environment, transparent exopolymer particles (TEP) are ubiquitous and abundant, playing a significant role in carbon cycling and the structuring of food webs. Previous studies have shown that phytoplankton, bacteria, and oysters contribute to the production of TEP through the release of exopolymers. However, little is known about other potential sources of TEP and TEP precursors, especially in coastal systems. It was hypothesized that suspension feeders contribute to the TEP pool in near-shore environments through the release of exopolymers in both the dissolved and particulate form, and tested these hypotheses in both laboratory and field experiments. In the laboratory, the production of TEP by several species of benthic suspension feeders (the blue mussel, Mytilus edulis; the bay scallop, Argopecten irradians; the slipper snail, Crepidula fornicata; and the solitary ascidians, Ciona intestinalis and Styela clava) was investigated from October to November 2002 and June 2003. Concentrations of TEP and DOC were determined by spectrophotometry after alcian blue staining and high-temperature, catalytic oxidation, respectively. Similar analyses were conducted on water samples from the field in July 2003, collected in close proximity to dense beds of mussels in the Poquonnock River, Connecticut, USA (41°19′ N, − 72°02′ W). Laboratory results indicated that actively-pumping blue mussels, bay scallops, slipper snails, and both species of solitary ascidians significantly enhanced TEP concentrations above background levels over a five-hour period. However, only the solitary tunicate S. clava significantly enhanced DOC concentrations above background levels over the same period of time. Field samples indicated that TEP and DOC concentrations were high in close proximity to dense beds of mussels. These results imply that a variety of benthic suspension feeders produce TEP during feeding activities which could lead to enhanced flocculation of organic matter and carbon deposition in near-shore waters.     

Dissolved organic matter (DOM) release by phytoplankton in the contemporary and future ocean

The partitioning of organic matter (OM) between dissolved and particulate phases is an important factor in determining the fate of organic carbon in the ocean. Dissolved organic matter (DOM) release by phytoplankton is a ubiquitous process, resulting in 2–50% of the carbon fixed by photosynthesis leaving the cell. This loss can be divided into two components: passive leakage by diffusion across the cell membrane and the active exudation of DOM into the surrounding environment. At present there is no method to distinguish whether DOM is released via leakage or exudation. Most explanations for exudation remain hypothetical; as while DOM release has been measured extensively, there has been relatively little work to determine why DOM is released. Further research is needed to determine the composition of the DOM released by phytoplankton and to link composition to phytoplankton physiological status and environmental conditions. For example, the causes and physiology of phytoplankton cell death are poorly understood, though cell death increases membrane permeability and presumably DOM release. Recent work has shown that phytoplankton interactions with bacteria are important in determining both the amount and composition of the DOM released. In response to increasing CO₂ in the atmosphere, climate change is creating increasingly stressful conditions for phytoplankton in the surface ocean, including relatively warm water, low pH, low nutrient supply and high light. As ocean physics and chemistry change, it is hypothesized that a greater proportion of primary production will be released directly by phytoplankton into the water as DOM. Changes in the partitioning of primary production between the dissolved and particulate phases will have bottom-up effects on ecosystem structure and function. There is a need for research to determine how these changes affect the fate of organic matter in the ocean, particularly the efficiency of the biological carbon pump.     

TRANSPARENT EXOPOLYMER PARTICLES FORMATION FROM CAPSULES OF ANABAENA SPIROIDES (CYANOBACTERIA) IN CULTURE1

We report the production of large numbers of transparent exopolymer particles (TEP) from polysaccharidic capsules of Anabaena spiroides Kleb. in cultures. Two biotic pathways of TEP formation were observed: (1) fragmentation of small portions of the capsules, which occurred throughout the growth phases; and (2) transformation of the whole polysaccharidic capsules into TEP, following cellular lyses in the aging culture. Photographic documentation of these processes was performed after staining small aliquots of the samples with Alcian Blue and negative staining with India ink. Concentrations of TEP were determined in distinct culture growth phases using semiquantitative Alcian Blue staining. Concentrations of TEP increased throughout the experimental time, while Alcian Blue remaining in solution decreased. Decreasing concentrations of chl a indicated cellular death, and by the end of the experiment, TEP formed by both pathways accumulate in the culture medium. These results show that virtually all dead chains of A. spiroides are transformed into TEP in the aged culture.     

海洋浮游植物与生物碳汇

系统描述了浮游植物与海洋碳汇相关的几个过程:初级生产、浮游植物沉降、浮游动物粪球打包沉降、经典食物链碳汇、溶解有机碳生产和转化、透明胞外聚合颗粒物(TEP)凝聚网,和CO₂分压升高(海水酸化)影响下浮游植物功能群转变及中国海可能的生物碳汇前景展望。提出海洋初级生产过程和TEP凝聚网过程是中国海生物碳汇的关键过程,而中国海的黄海中部及长江口区域是生物碳汇研究的重点区域,建议将硅藻及其碳汇过程作为今后研究的重点。     

海洋中的凝集网与透明胞外聚合颗粒物

透明胞外聚合颗粒物(TEP)是海洋中大量存在的能被爱尔新蓝(alcian blue)染色的由酸性多糖组成的透明胶状颗粒物,主要来源于浮游植物,由于其透明的特性而被长期忽视。TEP同时具有胶体和颗粒物的特性。作为胶体,TEP为细菌提供了栖息场所与降解基质,同时TEP可以吸收痕量元素,以改变这些元素的生物地球化学过程。作为颗粒物,TEP可以聚集并沉降,由于其高的碳含量,会在很大程度上影响海洋的碳通量。由于TEP可以被中型浮游动物所摄食,所以TEP可以连接并缩短微食物环和经典食物链,在海洋生态系统中起很重要的作用。介绍了TEP的定义、测量方法、来源、形成、及其与浮游植物的关系和其生态功能。     

Dissolved organic carbon and bacterial populations in the gelatinous surface microlayer of a Norwegian fjord mesocosm

The sea surface microlayer is the interfacial boundary layer between the marine environment and the troposphere. Surface microlayer samples were collected during a fjord mesocosm experiment to study microbial assemblage dynamics within the surface microlayer during a phytoplankton bloom. Transparent exopolymer particles were significantly enriched in the microlayer samples, supporting the concept of a gelatinous surface film. Dissolved organic carbon and bacterial cell numbers (determined by flow cytometry) were weakly enriched in the microlayer samples. However, the numbers of Bacteria 16S rRNA genes (determined by quantitative real-time PCR) were more variable, probably due to variable numbers of bacterial cells attached to particles. The enrichment of transparent exopolymer particles in the microlayer and the subsequent production of a gelatinous biofilm have implications on air–sea gas transfer and the partitioning of organic carbon in surface waters.     

Production of macroaggregates from dissolved exopolymeric substances (EPS) of bacterial and diatom origin

Exopolymeric substances (EPS) isolated from a pure culture of the marine bacterium Marinobacter sp. and the marine diatom Skeletonema costatum (axenic) were partially purified, chemically characterized and used as dissolved organic matter (DOM) for the production of macroaggregates. The role of organic particles such as transparent exopolymeric particles (TEP) and Coomassie stained particles (CSP) in the production of macroaggregates was experimentally assessed. Three experimental rolling tanks containing sterile medium with: (1) EPS, (2) EPS + live diatom cells and (3) EPS + killed bacteria, and three control tanks without any added EPS were used for macroaggregate production. Changes in abundance and average size of macroaggregates were monitored using image analysis, whereas TEP and CSP were enumerated microscopically. In the presence of microbial EPS, macroaggregates of a size of 23-35 mm(2) were produced. Aggregate size and abundance considerably varied with both time and source of EPS. No correlation was observed for macroaggregate size and abundance with either TEP or CSP. One-way ANOVA demonstrated significant differences in the variance of particle abundance and size in tanks having only EPS or EPS in combination with live diatom cells. Our data suggest that production of macroaggregates was influenced by polymer chemistry and surface properties of colliding particles, whereas TEP and CSP concentrations were influenced by molecular weight of EPS and the presence of growing cells. Interestingly, macroaggregates were formed in the near absence of TEP and CSP, highlighting the role of other unknown processes in the transformation of DOM to particulate organic matter (POM) in aquatic environments.     

Transparent exopolymer particles (TEP) and their potential effect on membrane biofouling

Transparent exopolymer particles (TEP) have been described as a class of particulate acidic polysaccharides, which are large, transparent organic particles, and commonly found in seawater, surface water, and wastewater. Due to their unique physicochemical characteristics, more and more attention has recently been given to the effects of TEP on membrane fouling. In this review, the characteristics and determination methods of TEP as well as its potential effect on membrane biofouling are discussed. It appears that the analytical methods for TEP available in the literature are still debatable, and there is room for further improvement. Nevertheless, evidence suggests that TEP might be involved in the development of membrane fouling, especially at the early stage of biofilm development on membranes.     

Aquatic polymers can drive pathogen transmission in coastal ecosystems

Gelatinous polymers including extracellular polymeric substances (EPSs) are fundamental to biophysical processes in aquatic habitats, including mediating aggregation processes and functioning as the matrix of biofilms. Yet insight into the impact of these sticky molecules on the environmental transmission of pathogens in the ocean is limited. We used the zoonotic parasite Toxoplasma gondii as a model to evaluate polymer-mediated mechanisms that promote transmission of terrestrially derived pathogens to marine fauna and humans. We show that transparent exopolymer particles, a particulate form of EPS, enhance T. gondii association with marine aggregates, material consumed by organisms otherwise unable to access micrometre-sized particles. Adhesion to EPS biofilms on macroalgae also captures T. gondii from the water, enabling uptake of pathogens by invertebrates that feed on kelp surfaces. We demonstrate the acquisition, concentration and retention of T. gondii by kelp-grazing snails, which can transmit T. gondii to threatened California sea otters. Results highlight novel mechanisms whereby aquatic polymers facilitate incorporation of pathogens into food webs via association with particle aggregates and biofilms. Identifying the critical role of invisible polymers in transmission of pathogens in the ocean represents a fundamental advance in understanding and mitigating the health impacts of coastal habitat pollution with contaminated runoff.     

Algae-bacteria interactions and their effects on aggregation and organic matter flux in the sea

Aggregation of algae, mainly of diatoms, is an important process in marine pelagic systems, often terminating phytoplankton blooms and leading to the sinking of particulate organic matter in the form of marine snow. This process has been studied extensively, but the specific role of heterotrophic bacteria has largely been neglected, mainly because field studies and most experimental work were performed under non-axenic conditions. We tested the hypothesis that algae-bacteria interactions are instrumental in aggregate dynamics and organic matter flux. A series of aggregation experiments has been carried out in rolling tanks with two marine diatoms typical of temperate regions (Skeletonema costatum and Thalassiosira rotula) in an axenic treatment and one inoculated with marine bacteria. Exponentially growing S. costatum and T. rotula exhibited distinctly different aggregation behavior. This was reflected by their strikingly different release of dissolved organic matter (DOM), transparent exopolymer particles (TEP) and protein-containing particles (CSP), as well as their bacterial biodegradability and recalcitrance. Cells of S. costatum aggregated only little and their bacterial colonization remained low. Dissolved organic matter, TEP and CSP released by this alga were largely consumed by free-living bacteria. In contrast, T. rotula aggregated rapidly and DOM, TEP and CSP released resisted bacterial consumption. Experiments conducted with T. rotula cultures in the stationary growth phase, however, showed rapid bacterial colonization and decomposition of algal cells. Our study highlights the importance of heterotrophic bacteria to control the development and aggregation of phytoplankton in marine systems.     

Marine bacterioplankton production of polysaccharidic and proteinaceous particles under different nutrient regimes

The influence of inorganic nutrient concentrations on the ability of bacterioplankton to produce and degrade polysaccharidic transparent exopolymer particles (TEPs) and proteinaceous Coomassie-stained particles (CSPs) was investigated in an 11-day experiment. The dynamics of these particles were followed in prefiltered (1 microm) northern Adriatic seawater enclosures enriched either with 1 microM orthophosphate (main limiting nutrient in this area), 10 microM ammonium or both orthophosphate and ammonium. These enclosures were referenced to a nonenriched control. A high potential for bacterial TEP and CSP production was observed (10(4) - 10(5) L(-1) for particles larger than 4 microm). In conditions of high orthophosphate concentration (either orthophosphate enriched or both orthophosphate and ammonium enriched), lower abundances and surface areas of CSPs were obtained, whereas TEP dynamics were more affected by unbalanced enrichments where only orthophosphate or ammonium was added. The impact of unbalanced nutrient ratios on TEPs was indicated by their higher abundance but low capacity for Alcian blue absorption, implying a change in their structure. Inorganic nutrient availability was thus proven to affect the bacterial potential for producing and degrading bacterially derived TEPs and CSPs.     

Abiotic stress induces apoptotic-like features in tobacco that is inhibited by expression of human Bcl-2

The shared features between plant and animal programmed cell death are becoming increasingly apparent. In this study, human Bcl-2, an anti-apoptotic member of the Bcl-2 family of cell death regulators, was stably expressed in tobacco. Previously, we have shown that such plants were resistant/tolerant to several necrotrophic fungal pathogens. In this study, we show that transgenic plants are protected by several lethal abiotic stresses including heat, cold, menadione and hydrogen peroxide. Importantly, wild type tobacco, exposed to these treatments, not only died but during the death process exhibited features associated with mammalian apoptosis including DNA laddering, fragmentation, and the development of apoptotic bodies. These features were not observed in viable transgenic tobacco. Thus, abiotic stress induced cell death in plants can be accompanied by apoptotic-like features that are inhibited by expression of Bcl-2. These observations add to the growing body of evidence indicating trans-kingdom conservation of programmed cell death mechanisms.     

Dynamics of transparent exopolymeric particles (TEP) production by Phaeocystis globosa under N- or P-limitation: a controlling factor of the retention/export balance

The concentration of transparent exopolymeric particles (TEP) was monitored during Phaeocystis globosa blooms that developed in mesocosms under different initial N:P ratios (from N- to P-limited conditions). TEP concentration was measured using the microscopic (TEP_micro, ppm) and the colorimetric (TEP_color, Xanthan equiv. L⁻¹) methods. TEP concentrations varied from 5 to >75 ppm and from 60 to >1500 μg Xanthan equiv. L⁻¹, and were relatively low until the mesocosms reached nutrient (either N or P) depletion and then increased abruptly. From the TEP_micro versus TEP_color concentrations comparison and from their relation to chlorophyll a concentrations, two phases for the dynamics of TEP production were identified: (1) production through active release of precursors during the growth phase of P. globosa — defined as TEP₁ — and their integration into the TEP pool through coagulation processes; (2) release of large TEP from the mucilaginous matrix of P. globosa colonies subsequent to disruption caused by nutrient depletion — defined as TEP₂ — and their direct integration into the TEP pool outside the constraint of coagulation. The formation of a multiorigin TEP pool during P. globosa blooms may have implications for the fate of the blooms, due to difference in TEP bioreactivity according to their source and to difference in timing and intensity of TEP₁ versus TEP₂ production according to N- or P-depletion. For P. globosa blooms developing under N-limiting conditions, the transition from the first source (i.e. TEP₁) to the second one (i.e. TEP₂) was a slow and continuous process. In contrast, the P. globosa bloom developing under P-limiting conditions showed the sudden formation of heavy mucous aggregates when P became depleted, that may have been caused by a massive release of TEP₂. Our study suggests that the nutrient regime may control the export vs. retention balance during P. globosa blooms, via production of a multiorigin TEP pool.     

Hypersensitive response, cell death and histochemical localisation of hydrogen peroxide in host and non-host seedlings infected with the downy mildew pathogen Sclerospora graminicola

Hypersensitive response, cell death and release of hydrogen peroxide as measures of host and non‐host defense mechanisms upon inoculation with the downy mildew pathogen Sclerospora graminicola were studied histochemically at the light microscopy level. The materials consisted of coleoptile tissues of the highly susceptible (cv. HB3), highly resistant (cv. IP18293) and induced resistant pearl millet host seedlings and non‐host sorghum (cv. SGMN10/8) and cotyledon of french bean (cv. S9). Resistance up to 80% protection against the downy mildew pathogen was induced in the highly susceptible HB3 cultivar of pearl millet by treating the seeds with 2% aqueous leaf extract of Datura metel for 3 h. Time course study with the pathogen inoculated highly resistant pearl millet cultivar revealed the appearance of hypersensitive response in 20% of seedlings as necrotic spots as early as 2 h after inoculation. In contrast, a similar reaction was observed in the highly susceptible pearl millet cultivar only 8 h after inoculation with the pathogen. In induced resistant seedlings, appearance of hypersensitive response was recorded 4 h after inoculation. Delayed hypersensitive response was observed in both the non‐host species at 10 h after inoculation. Hypersensitive response in the seedlings of the highly resistant pearl millet cultivar 24 h after inoculation showed 100% hypersensitive response, which was not observed in susceptible and non‐host species, although the induced resistant seedlings showed 90% hypersensitive response after that period of time. Cell death in the tissues of the test seedlings was also observed to change with time. Statistical analysis revealed that the tissues of highly resistant pearl millet seedlings required 2.9 h to attain 50% cell death. Tissues of induced resistant and highly susceptible pearl millet seedlings required 4.65 and 6.50 h respectively. In non‐hosts, 50% cell death was not recorded. Quantification of hydrogen peroxide in the tissue periplasmic spaces of the test seedlings revealed 2.94 h as the time required for 50% hydrogen peroxide accumulation in the tissues of highly resistant pearl millet seedlings. Tissues of induced resistant and highly susceptible pearl millet seedlings needed 3.76 and 5.5 h respectively. Fifty percent hydrogen peroxide localisation in non‐hosts could not be recorded. These results suggested the involvement of hydrogen peroxide, cell death and hypersensitive response in pearl millet host defense against S. graminicola.     

Extracellular Polymer Produced in the Presence of Copper Minerals

Abstract

A qualitative and semiquantitative assay was applied to estimate production of peripheral exopolymers by strains of bacteria isolated from flotation wastes during growth on copper sulphides and copper minerals wastes at neutral pH. Exopolymer production, expressed as percentage of Alcian Blue adsorption per μ g of cell protein, was the lowest on a complete organic medium and the highest on a nutrient-limited mineral medium. The exopolymer was highly resistant to acid hydrolysis and contained hydrocarbons, saturated and unsaturated fatty acids, aromatic acids and aromatic acid esters, which are well known as artificial surfactants and plasticizers. Carbohydrates appeared incidentally and in trace amount only. Chemical components of the exopolymer or chemically similar compounds also were found in black shale particles in the flotation wastes.