Alkaline phosphatase (AP) activity in marine and freshwater phytoplankton has been associated with phosphorus (P) limitation whereby the enzyme functions in the breakdown of exogenous organic P con~pounds to utilizable inorganic forms. Current enzyme assays to determine the P status of the phytoplankton measure only the AP activity of the whole community and do not yield information on ~ndiv~dual species. A new insoluble fluorogenic substrate for AP, termed ELF (EnzymeLabeled Fluorescence), yields a stable, highly fluorescent precipitate at the site of enzyme act~vity and thus has the capability to determine the P status of indimdual cells. In t h~s study, ELF was utilized for in situ detection and quantification of AP in marine phytoplankton cultures and a comparison was made between the insoluble ELF substrate and several soluble AP substrates [3-0-methylfluorescein phosphate (MFP), 3,6-fluorescein diphosphate (FDP) and Attophos]. Non-axenic batch cultures of Alexandrium fundyense, Arnphidln~um sp, and Isochrysis galbana were grown in different media types using orthophosphate as an inorganic source and sodium-glycerophosphate as an organic source, with final phosphate concentrations ranging from 38.3 to 3 p M (i.e. f/2, f/40, f/80, plus ambient P). Epifluorescence microscopy was used to determine if and where the cells were labeled with ELF, while flow cytometry was used to quantify the amount of ELF retained on individual cells. The detection of the soluble substrates utilized a multiwell fluorescence plate reader (CytofluorTh4). Only cells grown in low phosphate concentrations (f/40, f/80) exhibited the bright green fluorescence signal of the ELF precipItate. This signal was always observed for P-starved Amphidiniurn sp. and I galbana cells, but was seen in some A, fundyense cells only during the late stationary phase. Cells grown in high phosphate concentrations (i.e. at f/2 levels) showed no ELF fluorescence. Slightly positive soluble substrate assays suggest that these species may have produced small amounts of AP constitutively that were not detected with the precipitable substrate. Similar results were obtained when the cultures were analyzed by flow cytometry. Except for A. fundyense, cells grown In low phosphate concentrations showed high ELF fluorescence. However, no positive ELF fluorescence was detected with the Cytofluor for all 3 species due to lack of instrument sensitivity. Comparable analysis using the soluble substrates MFP, FDP, and Att~phos-l-~' on the Cytofluor showed little activity for A , fundyense, but high fluorescence for P-starved Amphldiniun? sp. and I. galbana. Insoluble ELF thus provides a means to detect and quantify AP in individual cells using visual observations or flow cytometry. This technique offers a new level of resolution and sensitivity at the single cell level that can provide insights into the P nutrition of phytoplankton and other microorganisms in natural waters.
The correlation between water physicochemical parameters and eukaryotic benthic composition was examined in Río Tinto. Principal component analysis showed a high inverse relationship between pH and most of the heavy metals analyzed as well as Dunaliella sp., while Chlamydomonas sp. abundance was positively related. Zn, Cu, and Ni clustered together and showed a strong inverse correlation with the diversity coefficient and most of the species analyzed. These eukaryotic communities seem to be more influenced by the presence of heavy metals than by the pH.Natural extreme acidic rivers are scarce worldwide (18). Extreme acidic environments, characterized by a pH of Ͻ3, are often the consequence of anthropogenic influences (e.g., mining activity or acid rain) (14). Thus, most ecological studies of acidic waters have been focused on environments affected by human activity. In addition, most of the information available about acidophilic communities in aquatic environments is focused on bacterial communities, although microbial eukaryotes are also present and could also play a critical role in these places. There have been few reports on eukaryotes, and most of them are related to acid mine drainages instead of naturally acidic locations (2,3,16). Moreover, these studies are often carried out in lakes, probably because of the difficulty in obtaining integrated representative samples in rivers.In this regard, the Río Tinto (southwestern Spain), a 92-kmlong river, is one of the most extensive examples of a naturally extreme acidic environment. The river springs up in the core of the Iberian Pyritic Belt, one of the largest bodies of iron and copper sulfide deposits in the world (5). Ferric iron and sulfuric acid are the most common components found in this acidic environment, establishing a buffer system at pH values of approximately 2.3. Ferric iron is produced by the metabolism of ironoxidizing microorganisms, which are very active in the aerobic part of the river; sulfuric acid originates from sulfides by chemical oxidation or the activity of sulfur-oxidizing microorganisms, depending on the sulfide mineral substrate (15). The result is a strongly acidic solution of ferric iron which brings into solution other heavy metals, increasing their concentrations in relation to neighboring rivers with higher pH (12).It is usually assumed that high metal concentrations in acidic habitats limit eukaryotic growth and diversity due to their toxicity. It has been also proposed that metal hydroxide deposition could change the physicochemical conditions of surfaces, resulting in a reduction of epiphytic growth on rocks (8). However, what makes Río Tinto a unique acidic extreme environment is that eukaryotic organisms are the principal contributors of biomass in the river, over 65% of the total biomass, as well as the unexpected degree of eukaryotic diversity found in its waters (1, 21, 28). Members of the Bacillariophyta, Chlorophyta, and Euglenophyta phyla as well as ciliates, cercomonads, amoebae, stramenopiles, fungi, and yeast h...
Understanding biotic versus abiotic forces that shape community structure is a fundamental aim of microbial ecology. The acidic and heavy metal extreme Río Tinto (RT) in southwestern Spain provides a rare opportunity to conduct an ecosystem-wide biodiversity inventory at the level of all three domains of life, because diversity there is low and almost exclusively microbial. Despite improvements in high-throughput DNA sequencing, environmental biodiversity studies that use molecular metrics and consider entire ecosystems are rare. These studies can be prohibitively expensive if domains are considered separately, and differences in copy number of eukaryotic ribosomal RNA genes can bias estimates of relative abundances of phylotypes recovered. In this study we have overcome these barriers (1) by targeting all three domains in a single polymerase chain reaction amplification and (2) by using a replicated sampling design that allows for incidencebased methods to extract measures of richness and carry out downstream analyses that address community structuring effects. Our work showed that combined bacterial and archaeal richness is an order of magnitude higher than eukaryotic richness. We also found that eukaryotic richness was highest at the most extreme sites, whereas combined bacterial and archaeal richness was highest at less extreme sites. Quantitative community phylogenetics showed abiotic forces to be primarily responsible for shaping the RT community structure. Canonical correspondence analysis revealed co-occurrence of obligate symbionts and their putative hosts that may contribute to biotic forces shaping community structure and may further provide a possible mechanism for persistence of certain low-abundance bacteria encountered in the RT.
Due to its highly metalliferous waters and low pH, the Rio Tinto has shown its potential for modelling both acid mine drainage systems and biohydrometallurgical operations. Most geomicrobiological studies of these systems have focused on the oxic water column. A sequence-based approach in combination with in situ detection techniques enabled us to examine the composition and structure of the microbial communities associated with the suboxic and anoxic sediments along the river course and to compare them with the planktonic communities inhabiting the water column. The results obtained with the different approaches were consistent and revealed some major patterns: higher cell density and higher richness (75 vs. 48 operational taxonomic units) in the sediments than in the water column. The microbial communities were related but the river sediments appear to be enriched in certain populations, some of which have not previously been reported in the Rio Tinto basin. The differences detected between sampling stations along the river correlate with certain environmental parameters (e.g. iron concentration gradient). The biological and geochemical data show the importance of the sediments as representing a phase of particular high diversity, probably related to key metabolic processes within both the iron and the sulfur cycles.
An in situ colonization assay was performed to study the early stages of biofilm formation in Rio Tinto (SW, Spain), an extremely acidic environment (pH ca. 2). Eukaryotic assemblages were monitored at monthly intervals for 1 year. Diversity, colonization rates, and seasonal variations were analyzed. Structural features of naturally grown biofilms were explored by light and scanning electron microscopy in backscattered electron mode. A total of 14 taxa were recognized as constituents of the eukaryotic assemblages. The eukaryotic communities were dissimilar at the different sampling sites. The lowest diversity was found at the most extreme locations, in terms of pH and heavy metal concentrations. The biofilms were mainly formed by species from the genera Dunaliella and Cyanidium. Two genera of filamentous algae, Zygnemopsis and Klebsormidium, were principally responsible for the variability in the cell number throughout the year. These species appear in June to decrease almost completely between October and November. In contrast, the number of heterotrophic flagellates and ciliates remained constant throughout the year. The microcolonization sequence showed an initial accumulation of amorphous particles composed of bacteria and inorganic grains of minerals. By the end of the second month, the organic matrix was also populated by fungi, bacteria, and a few eukaryotic heterotrophs such as amoebae and small flagellates. Diatoms only showed significant colonization in regions where mycelial matrices were first established. Flagellated green algae such as Dunaliella or Chlamydomonas as well as Euglena were also present at the very beginning of the biofilm development, although in low numbers (<100 cells cm(-2)). After the flagellated cells, sessile species of algae such Chlorella or Cyanidium appeared. Filamentous algae were the last species to colonize the biofilms. Most of the naturally grown biofilms were found to be structures composed of different species organized in different layers separated, probably by extracellular polymeric substances, although more analysis should be done in this regard. The possible implications of the biofilm structure in the adaptation to this extreme habitat are discussed.
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