Use of electrospray ionization (ESI) mass spectrometry to investigate complex dissolved organic matter (DOM) and its potential applications in phytoplankton research

Organic nutrients are one of many factors considered to be important in the growth and proliferation of phytoplankton including many species that cause harmful algal blooms (HABs). Several studies have investigated the effects of known organic compounds on phytoplankton growth, however, the role of natural dissolved organic matter (DOM) in phytoplankton nutrition remains understudied at the compound level. This lack of research is due in part to analytical limitations for the characterization of DOM compounds. Electrospray ionization (ESI) mass spectrometry (MS) provides an unprecedented level of chemical information on thousands of organic compounds that comprise the bulk DOM pool. In this paper we provide a brief overview of some of the benefits and caveats of using ESI to investigate DOM in natural freshwater and marine systems and show an example of ESI-MS DOM characterization for a natural bloom of the raphidophyte Chattonella cf. verruculosa.     

Influence of dissolved organic matter from terrestrial origin on the changes of dinoflagellate species composition in the Gulf of Riga,Baltic Sea

Hydrobiologia - A mesocosm experiment was used to investigate the effect of terrestrial-origin dissolved organic matter (DOM) on the development of dinoflagellates in natural summer phytoplankton...     

Seasonal Response of Stream Biofilm Communities to Dissolved Organic Matter and Nutrient Enrichments

Dissolved organic matter (DOM) and inorganic nutrients may affect microbial communities in streams, but little is known about the impact of these factors on specific taxa within bacterial assemblages in biofilms. In this study, nutrient diffusing artificial substrates were used to examine bacterial responses to DOM (i.e., glucose, leaf leachate, and algal exudates) and inorganic nutrients (nitrate and phosphate singly and in combination). Artificial substrates were deployed for five seasons, from summer 2002 to summer 2003, in a northeastern Ohio stream. Differences were observed in the responses of bacterial taxa examined to various DOM and inorganic nutrient treatments, and the response patterns varied seasonally, indicating that resources that limit the bacterial communities change over time. Overall, the greatest responses were to labile, low-molecular-weight DOM (i.e., glucose) at times when chlorophyll a concentrations were low due to scouring during significant storm events. Different types of DOM and inorganic nutrients induced various responses among bacterial taxa in the biofilms examined, and these responses would not have been apparent if they were examined at the community level or if seasonal changes were not taken into account.     

Community shifts of actively growing lake bacteria after N-acetyl-glucosamine addition: improving the BrdU-FACS method

In aquatic environments, community dynamics of bacteria, especially actively growing bacteria (AGB), are tightly linked with dissolved organic matter (DOM) quantity and quality. We analyzed the community dynamics of DNA-synthesizing and accordingly AGB by linking an improved bromodeoxyuridine immunocytochemistry approach with fluorescence-activated cell sorting (BrdU-FACS). FACS-sorted cells of even oligotrophic ecosystems in winter were characterized by 16S rRNA gene analysis. In incubation experiments, we examined community shifts of AGB in response to the addition of N-acetyl-glucosamine (NAG), one of the most abundant aminosugars in aquatic systems. Our improved BrdU-FACS analysis revealed that AGB winter communities of oligotrophic Lake Stechlin (northeastern Germany) substantially differ from those of total bacteria and consist of Alpha-, Beta-, Gamma-, Deltaproteobacteria, Actinobacteria, Candidatus OP10 and Chloroflexi. AGB populations with different BrdU-fluorescence intensities and cell sizes represented different phylotypes suggesting that single-cell growth potential varies at the taxon level. NAG incubation experiments demonstrated that a variety of widespread taxa related to Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Planctomycetes, Spirochaetes, Verrucomicrobia and Chloroflexi actively grow in the presence of NAG. The BrdU-FACS approach enables detailed phylogenetic studies of AGB and, thus, to identify those phylotypes which are potential key players in aquatic DOM cycling.     

Bacterioplankton Community Shifts in an Arctic Lake Correlate with Seasonal Changes in Organic Matter Source

Seasonal shifts in bacterioplankton community composition in Toolik Lake, a tundra lake on the North Slope of Alaska, were related to shifts in the source (terrestrial versus phytoplankton) and lability of dissolved organic matter (DOM). A shift in community composition, measured by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes, occurred at 4°C in near-surface waters beneath seasonal ice and snow cover in spring. This shift was associated with an annual peak in bacterial productivity ([¹⁴C]leucine incorporation) driven by the large influx of labile terrestrial DOM associated with snow meltwater. A second shift occurred after the flux of terrestrial DOM had ended in early summer as ice left the lake and as the phytoplankton community developed. Bacterioplankton communities were composed of persistent populations present throughout the year and transient populations that appeared and disappeared. Most of the transient populations could be divided into those that were advected into the lake with terrestrial DOM in spring and those that grew up from low concentrations during the development of the phytoplankton community in early summer. Sequencing of DNA in DGGE bands demonstrated that most bands represented single ribotypes and that matching bands from different samples represented identical ribotypes. Bacteria were identified as members of globally distributed freshwater phylogenetic clusters within the α- and β-Proteobacteria, the Cytophaga-Flavobacteria-Bacteroides group, and the Actinobacteria.     

Phytoplankton distribution in the upwelling areas of the Moroccan Atlantic coast localized between 32°30'N and 24°N

Marine phytoplankton was studied in January and July 2002 along of four transects: 32°30′N, 29°N, 24°30′N and 24°N. A total of 142 taxa were recorded in this area. The maximal specific richness, both in summer and in winter, was registered around Dakhla (24°N). The global spatiotemporal variability of species richness and specific diversity showed that the most structured and species-rich populations are situated in the coastal areas. The vertical variation of the two parameters showed homogeneity between depth levels. The maximal phytoplanktonic densities are recorded in the southern transects due to the permanent upwelling activity in the southern Atlantic coast. The bathymetric distribution of densities was more heterogeneous during summer, characterized by a high intensity of upwelling. The species Thalassionema nitzschioides, Asterionellopsis glacialis, Melosira, Chaetoceros and Leptocylindrus minimus are indicators of upwelling.     

Effects of Seasonal Upwelling on Inorganic and Organic Matter Dynamics in the Water Column of Eastern Pacific Coral Reefs

The Gulf of Papagayo at the northern Pacific coast of Costa Rica experiences pronounced seasonal changes in water parameters caused by wind-driven coastal upwelling. While remote sensing and open water sampling already described the physical nature of this upwelling, the spatial and temporal effects on key parameters and processes in the water column have not been investigated yet, although being highly relevant for coral reef functioning. The present study investigated a range of water parameters on two coral reefs with different exposure to upwelling (Matapalo and Bajo Rojo) in a weekly to monthly resolution over one year (May 2013 to April 2014). Based on air temperature, wind speed and water temperature, three time clusters were defined: a) May to November 2013 without upwelling, b) December 2013 to April 2014 with moderate upwelling, punctuated by c) extreme upwelling events in February, March and April 2014. During upwelling peaks, water temperatures decreased by 7°C (Matapalo) and 9°C (Bajo Rojo) to minima of 20.1 and 15.3°C respectively, while phosphate, ammonia and nitrate concentrations increased 3 to 15-fold to maxima of 1.3 μmol PO₄ ^3- L^-1, 3.0 μmol NH₄ ⁺ L^-1 and 9.7 μmol NO₃ ^- L^-1. This increased availability of nutrients triggered several successive phytoplankton blooms as indicated by 3- (Matapalo) and 6-fold (Bajo Rojo) increases in chlorophyll a concentrations. Particulate organic carbon and nitrogen (POC and PON) increased by 40 and 70% respectively from February to April 2014. Dissolved organic carbon (DOC) increased by 70% in December and stayed elevated for at least 4 months, indicating high organic matter release by primary producers. Such strong cascading effects of upwelling on organic matter dynamics on coral reefs have not been reported previously, although likely impacting many reefs in comparable upwelling systems.     

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.     

Effects of nutrients and dissolved organic matter on the response of phytoplankton to ultraviolet radiation: experimental comparison in spring versus summer

The effects of nutrients and dissolved organic matter (DOM) on the response of phytoplankton community structure to ultraviolet radiation (UVR) was studied using natural phytoplankton assemblages from Lake Giles (Northeastern Pennsylvania), a temperate, oligotrophic, highly UVR-transparent lake. Microcosm experiments were conducted in 1-l bags in the spring and summer. A factorial design was used, with two UVR treatments (ambient and reduced), two nutrient treatments (control with no nutrients added, and nitrogen and phosphorus addition together), and two DOM treatments (control of 1 mg l⁻¹ and doubled). In April, UVR affected the overall phytoplankton community structure, causing a shift in the dominant species. Significant interactive effects of UVR × nutrients and UVR × DOM were found on total phytoplankton biovolumes. In July, all taxa responded positively to the N + P addition, and were affected differentially by the UVR treatments. The initial communities varied in April and July, but Synura sp. and Chroomonas sp. were present in both seasons. Synura sp. responded positively to the addition of DOM in April and the reduction of UVR in July. Chroomonas sp. responded positively to the reduction of UVR in April and the addition of nutrients in July. The differential sensitivity of these two species suggests that changing environmental factors between spring and summer promoted differences in the relative importance of UVR in changing phytoplankton community structure. Handling editor: Luigi Naselli-Flores     

Cell-specific beta-N-acetylglucosaminidase activity in cultures and field populations of eukaryotic marine phytoplankton.

It is widely appreciated that eukaryotic marine phytoplankton can hydrolyze a variety of compounds within the dissolved organic matter (DOM) pool in marine environments. Herein, cultures and field populations of marine phytoplankton were assayed for beta-N-acetylglucosaminidase activity, a terminal enzyme of chitin degradation. A traditional bulk assay, which can assess hydrolytic rate, but is not cell-specific, was complemented with a cell-specific assay that images the activity associated with single cells using an enzyme labeled fluorescence (ELF) substrate. beta-N-acetylglucosaminidase activity was widespread across various taxa of marine phytoplankton, and activity was observed both under controlled culture conditions and in field populations. The number of cells with enzyme activity varied with the nutritional physiology of the test species in three of the 17 cultures tested. In these three cases the number of cells with activity in the low nutrient medium was higher than in nutrient replete medium. Taken together, these data suggest that a broad group of marine phytoplankton may be a relevant part of chitin-like DOM degradation and should be incorporated into conceptual models of chitin cycling in marine systems.     

Phytoplankton distribution patterns in the northwestern Sargasso Sea revealed by small subunit rRNA genes from plastids

Phytoplankton species vary in their physiological properties, and are expected to respond differently to seasonal changes in water column conditions. To assess these varying distribution patterns, we used 412 samples collected monthly over 12 years (1991–2004) at the Bermuda Atlantic Time-Series Study site, located in the northwestern Sargasso Sea. We measured plastid 16S ribosomal RNA gene abundances with a terminal restriction fragment length polymorphism approach and identified distribution patterns for members of the Prymnesiophyceae, Pelagophyceae, Chrysophyceae, Cryptophyceae, Bacillariophyceae and Prasinophyceae. The analysis revealed dynamic bloom patterns by these phytoplankton taxa that begin early in the year, when the mixed layer is deep. Previously, unreported open-ocean prasinophyte blooms dominated the plastid gene signal during convective mixing events. Quantitative PCR confirmed the blooms and transitions of Bathycoccus, Micromonas and Ostreococcus populations. In contrast, taxa belonging to the pelagophytes and chrysophytes, as well as cryptophytes, reached annual peaks during mixed layer shoaling, while Bacillariophyceae (diatoms) were observed only episodically in the 12-year record. Prymnesiophytes dominated the integrated plastid gene signal. They were abundant throughout the water column before mixing events, but persisted in the deep chlorophyll maximum during stratified conditions. Various models have been used to describe mechanisms that drive vernal phytoplankton blooms in temperate seas. The range of taxon-specific bloom patterns observed here indicates that different ‘spring bloom'' models can aptly describe the behavior of different phytoplankton taxa at a single geographical location. These findings provide insight into the subdivision of niche space by phytoplankton and may lead to improved predictions of phytoplankton responses to changes in ocean conditions.     

Host-Specificity and Dynamics in Bacterial Communities Associated with Bloom-Forming Freshwater Phytoplankton

Many freshwater phytoplankton species have the potential to form transient nuisance blooms that affect water quality and other aquatic biota. Heterotrophic bacteria can influence such blooms via nutrient regeneration but also via antagonism and other biotic interactions. We studied the composition of bacterial communities associated with three bloom-forming freshwater phytoplankton species, the diatom Aulacoseira granulata and the cyanobacteria Microcystis aeruginosa and Cylindrospermopsis raciborskii. Experimental cultures incubated with and without lake bacteria were sampled in three different growth phases and bacterial community composition was assessed by 454-Pyrosequencing of 16S rRNA gene amplicons. Betaproteobacteria were dominant in all cultures inoculated with lake bacteria, but decreased during the experiment. In contrast, Alphaproteobacteria, which made up the second most abundant class of bacteria, increased overall during the course of the experiment. Other bacterial classes responded in contrasting ways to the experimental incubations causing significantly different bacterial communities to develop in response to host phytoplankton species, growth phase and between attached and free-living fractions. Differences in bacterial community composition between cyanobacteria and diatom cultures were greater than between the two cyanobacteria. Despite the significance, major differences between phytoplankton cultures were in the proportion of the OTUs rather than in the absence or presence of specific taxa. Different phytoplankton species favoring different bacterial communities may have important consequences for the fate of organic matter in systems where these bloom forming species occur. The dynamics and development of transient blooms may also be affected as bacterial communities seem to influence phytoplankton species growth in contrasting ways.     

Demonstration of extraction and PCR amplification of DNA from phytoplankton of lakes with high humic acid content

Lakes with high levels of dissolved organic matter are common in northern temperate regions. These habitats are sensitive to environmental and climate change, but the molecular ecology and eco-physiology of their phytoplankton have been under studied, in part because the DOM interferes with molecular and biochemical analyses of these populations. We developed a procedure which uses polyvinylpyrrolidone to adsorb interfering material during the extraction of nucleic acids from these populations. This procedure does not depend on enzymatic lysis and yields DNA of sufficient quality to serve as a template for PCR amplification of specific genetic sequences. In particular, we detected fragments of the conserved rbcL gene encoding the large subunit of the Rubisco enzyme from the phytoplankton of lakes with high DOM. Our extraction procedure may be useful to other workers studying similar populations.     

Uncoupling of Bacterioplankton and Phytoplankton Production in Fresh Waters Is Affected by Inorganic Nutrient Limitation

Pelagic bacterial production is often positively correlated, or coupled, with primary production through utilization of autotrophically produced dissolved organic carbon. Recent studies indicate that inorganic N or P can directly limit both bacterial and phytoplanktonic growth. Our mesocosm experiments, with whole communities from mesotrophic Calder Lake, test whether this apparent bacterial-algal coupling may be the result of independent responses to limiting inorganic nutrients. In systems without N additions, numbers of bacteria but not phytoplankton increased 2- to 2.5-fold in response to P fertilization (0 to 2.0 μmol of P per liter); this resulted in uncoupled production patterns. In systems supplemented with 10 μmol of NH₄NO₃ per liter, P addition resulted in up to threefold increases in bacteria and two- to fivefold increases in total phytoplankton biomass (close coupling). P limitation of pelagic bacteria occurred independently of phytoplankton dynamics, and regressions between bacterial abundance and phytoplankton chlorophyll a were nonsignificant in all systems without added N. We describe a useful and simple coupling index which predicts that shifts in phytoplankton and bacterioplankton growth will be unrelated (Δ bacteria/Δ phytoplankton → either + ∞ or - ∞) in systems with inorganic N/P (molar) ratios of <~40. In systems with higher N/P ratios (>40), the coupling index will approach 1.0 and close coupling between bacteria and phytoplankton is predicted to occur.     

Size‐spectrum based differential response of phytoplankton to nutrient and iron‐organic matter combinations in microcosm experiments in a Chilean Patagonian Fjord

The Patagonian fjords have been recognized as a major region of relatively high primary productivity systems during spring–summer bloom periods, where iron‐organic matter forms may be essential complexes involved in key growth processes connected to the carbon and nitrogen cycles. We used two dissolved organic matter (DOM) types, marine polysaccharide and siderophore, as a model to understand how they affect the bioavailability of Fe to phytoplankton and bacteria and to assess their ecological role in fjord systems. A 10‐day microcosm study was performed in the Comau Fjord during summer conditions (March 2012). Pico‐, nano‐, and microphytoplankton abundance, total chlorophyll‐a and bacteria abundance, and bacterial secondary production estimates were analyzed in five treatments: (i) control (no additions), (ii) only nutrients (NUT: PO₄, NO₃, Si), (iii) nutrients + Fe(II), (iv) polysaccharide (natural diatoms extracted: 1–3 beta Glucan), and (v) Hexandentate Desferroxiamine B (DFB, siderophore). Our results showed that while DFB reduced Fe bioavailability for almost all phytoplankton assemblages in the fjord, polysaccharide did not have effects on the iron bioavailability. At Nutrients + Fe and Polysaccharide treatments, chlorophyll‐a concentration abruptly increased from 0.9 to 20 mg m^?3 during the first 4–6 days of the experimental period. Remarkably, at the Nutrients + Fe treatment, the development of the bloom was accompanied by markedly high abundances of Synechococcus, picoeukaryotes, and autotrophic nanoflagellates within the first 4 days of the experiment. Our study indicated that small plankton (phytoplankton <20 μm and bacteria) were the first to respond to dissolved Nutrients + Fe compared to large sized micro‐phytoplankton cells (>20 μm). This could be at least partially attributed to biological utilization of Fe (2 to 3 nM) by <20 μm phytoplankton and bacteria through the interaction with organic ligands released by bacteria that eventually could increase solubility of the Fe dissolved fraction thus having a positive effect on the small‐sized phytoplankton community.     

Transporter genes expressed by coastal bacterioplankton in response to dissolved organic carbon

Coastal ocean bacterioplankton control the flow of dissolved organic carbon (DOC) from terrestrial and oceanic sources into the marine food web, and regulate the release of inorganic carbon to atmospheric and offshore reservoirs. While the fate of the chemically complex coastal DOC reservoir has long been recognized as a critical feature of the global carbon budget, it has been problematic to identify both the compounds that serve as major conduits for carbon flux and the roles of individual bacterioplankton taxa in mediating that flux. Here we analyse random libraries of expressed genes from a coastal bacterial community to identify sequences representing DOC‐transporting proteins. Predicted substrates of expressed transporter genes indicated that carboxylic acids, compatible solutes, polyamines and lipids may be key components of the biologically labile DOC pool in coastal waters, in addition to canonical bacterial substrates such as amino acids, oligopeptides and carbohydrates. Half of the expressed DOC transporter sequences in this coastal ocean appeared to originate from just eight taxa: Roseobacter, SAR11, Flavobacteriales and five orders of γ‐Proteobacteria. While all major taxa expressed transporter genes for some DOC components (e.g. amino acids), there were indications of specialization within the bacterioplankton community for others (e.g. carbohydrates, carboxylic acids and polyamines). Experimental manipulations of the natural DOC pool that increased the concentration of phytoplankton‐ or vascular plant‐derived compounds invoked a readily measured response in bacterial transporter gene expression. This highly resolved view of the potential for carbon flux into heterotrophic bacterioplankton cells identifies possible bioreactive components of the coastal DOC pool and highlights differing ecological roles in carbon turnover for the resident bacterial taxa.     

A large molecular size fraction of riverine high molecular weight dissolved organic matter (HMW DOM) stimulates growth of the harmful dinoflagellate Alexandrium minutum

An increase in the concentration of riverine dissolved organic matter (DOM) has been observed during the last decades, and this material can stimulate marine plankton in coastal waters with significant freshwater input. We studied the effect of two size fractions of riverine high molecular weight dissolved organic matter (HMW DOM), isolated with tangential ultrafiltration, on the harmful dinoflagellate Alexandrium minutum and a natural isolate of marine bacteria under laboratory conditions. Both A. minutum and bacteria grew significantly better with the low MW DOM compared to both the high MW DOM fraction and controls (no DOM additions). This experiment demonstrates that the harmful algae A. minutum and bacteria benefit from larger molecules of river HMW DOM, and highlights the potential of A. minutum to utilize organic nitrogen from large DOM molecules. This ability may enhance their likelihood of success in estuaries/costal waters with a humic rich freshwater input, especially when the relative amount of large molecules within DOM is more pronounced.