Investigation of an Iron-Oxidizing Microbial Mat Community Located near Aarhus, Denmark: Field Studies

We investigated the microbial community that developed at an iron seep where anoxic groundwater containing up to 250 μM Fe²⁺ flowed out of a rock wall and dense, mat-like aggregations of ferric hydroxides formed at the oxic-anoxic interface. In situ analysis with oxygen microelectrodes revealed that the oxygen concentrations in the mat were rarely more than 50% of air saturation and that the oxygen penetration depth was quite variable, ranging from <0.05 cm to several centimeters. The bulk pH of the mat ranged from 7.1 to 7.6. There appeared to be a correlation between the flow rates at different subsites of the mat and the morphotypes of the microorganisms and Fe oxides that developed. In subsites with low flow rates (<2 ml/s), the iron-encrusted sheaths of Leptothrix ochracea predominated. Miniature cores revealed that the top few millimeters of the mat consisted primarily of L. ochracea sheaths, only about 7% of which contained filaments of cells. Deeper in the mat, large particulate oxides developed, which were often heavily colonized by unicellular bacteria that were made visible by staining with acridine orange. Direct cell counts revealed that the number of bacteria increased from approximately 10⁸ to 10⁹ cells per cm³ and the total iron concentration increased from approximately 0.5 to 3 mmol/cm³ with depth in the mat. Primarily because of the growth of L. ochracea, the mat could accrete at rates of up to 3.1 mm/day at these subsites. The iron-encrusted stalks of Gallionella spp. prevailed in localized zones of the same low-flow-rate subsites, usually close to where the source water emanated from the wall. These latter zones had the lowest O₂ concentrations (<10% of the ambient concentration), confirming the microaerobic nature of Gallionella spp. In subsites with high flow rates (>6 ml/s) particulate Fe oxides were dominant; direct counts revealed that up to 10⁹ cells of primarily unicellular bacteria per cm³ were associated with these particulate oxides. These zones exhibited little vertical stratification in either the number of cells or iron concentration. Finally, mat samples incubated anaerobically in the presence of acetate or succinate exhibited significant potential for iron reduction, suggesting the possibility that a localized iron cycle could occur within the mat community.     

Community Composition of a Hypersaline Endoevaporitic Microbial Mat

A hypersaline, endoevaporitic microbial community in Eilat, Israel, was studied by microscopy and by PCR amplification of genes for 16S rRNA from different layers. In terms of biomass, the oxygenic layers of the community were dominated by Cyanobacteria of the Halothece, Spirulina, and Phormidium types, but cell counts (based on 4′,6′-diamidino-2-phenylindole staining) and molecular surveys (clone libraries of PCR-amplified genes for 16S rRNA) showed that oxygenic phototrophs were outnumbered by the other constituents of the community, including chemotrophs and anoxygenic phototrophs. Bacterial clone libraries were dominated by phylotypes affiliated with the Bacteroidetes group and both photo- and chemotrophic groups of α-proteobacteria. Green filaments related to the Chloroflexi were less abundant than reported from hypersaline microbial mats growing at lower salinities and were only detected in the deepest part of the anoxygenic phototrophic zone. Also detected were nonphototrophic γ- and δ-proteobacteria, Planctomycetes, the TM6 group, Firmicutes, and Spirochetes. Several of the phylotypes showed a distinct vertical distribution in the crust, suggesting specific adaptations to the presence or absence of oxygen and light. Archaea were less abundant than Bacteria, their diversity was lower, and the community was less stratified. Detected archaeal groups included organisms affiliated with the Methanosarcinales, the Halobacteriales, and uncultured groups of Euryarchaeota.     

Microbial Dynamics of an Epilithic Mat Community in a High Alpine Stream

Studies were conducted to examine interrelationships between the heterotrophic and phototrophic populations within an epilithic community in the outlet stream of a high alpine lake. Levels of nitrates, phosphates, and total organic compounds in the lake were consistently near the lower limits of detectability. Microscopic examination of the community by phase-contrast light microscopy and scanning electron microscopy revealed diatoms, filamentous algae, and bacteria embedded within a dense gelatinous matrix. Chlorophyll a and primary productivity measurements had peak values in early August, with subsequent declines. Bacterial heterotrophic activity, as measured by V_max, turnover rate, and relative activity, increased significantly as the phototrophic community declined. This trend in heterotrophic activity was not accompanied by an increase in total bacterial numbers as determined by epi-illuminated fluorescence microscopy. These results suggest that the phototrophic community responded to changes in, or interactions among, various chemical and physical factors throughout the study period. The catabolic activity of the sessile bacteria appeared to be positively influenced by changes in the mat environment resulting from the decline of the phototrophic populations.     

Algal Species and Light Microenvironment in a Low-pH, Geothermal Microbial Mat Community

Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to <1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. A Cyanidioschyzon merolae sequence was predominant at temperatures of ≥49°C. A Chlorella protothecoides var. acidicola sequence and a Paradoxia multisita-like sequence were predominant at temperatures of ≤39°C.     

Biogeochemistry of an Iron-Rich Hypersaline Microbial Mat (Camargue, France)

In situ microsensor measurements were combined with biogeochemical methods to determine oxygen, sulfur, and carbon cycling in microbial mats growing in a solar saltern (Salin-de-Giraud, France). Sulfate reduction rates closely followed the daily temperature changes and were highest during the day at 25°C and lowest during the night at 11°C, most probably fueled by direct substrate interactions between cyanobacteria and sulfate-reducing bacteria. Sulfate reduction was the major mineralization process during the night and the contribution of aerobic respiration to nighttime DIC production decreased. This decrease of aerobic respiration led to an increasing contribution of sulfide (and iron) oxidation to nighttime O₂ consumption. A peak of elemental sulfur in a layer of high sulfate reduction at low sulfide concentration underneath the oxic zone indicated anoxygenic photosynthesis and/or sulfide oxidation by iron, which strongly contributed to sulfide consumption. We found a significant internal carbon cycling in the mat, and sulfate reduction directly supplied DIC for photosynthesis. The mats were characterized by a high iron content of 56 mol Fe cm^–3, and iron cycling strongly controlled the sulfur cycle in the mat. This included sulfide precipitation resulting in high FeS contents with depth, and reactions of iron oxides with sulfide, especially after sunset, leading to a pronounced gap between oxygen and sulfide gradients and an unusual persistence of a pH peak in the uppermost mat layer until midnight.     

Community Structure,Geochemical Characteristics and Mineralogy of a Hypersaline Microbial Mat,Cabo Rojo,PR

Seasonal variations in precipitation changed the community composition and microbial activity in a hypersaline, tropical microbial mat, in Cabo Rojo, PR. Using a combination of dissection, light, and transmission electron microscopy, terminal restriction fragment length polymorphism (T-RFLP), in situ microelectrode studies, and ³⁵ S isotope incubations, we documented the major differences between wet and dry seasons. During the wet season (precipitation 177 mm), cyanobacterial (green layer) and anoxyphototrophic (pink layer) communities, as well as the black FeS layer were well-developed, and T-RFLP patterns indicated a diverse community. The rate of oxygenic photosynthesis was 49 μ M min ^{? 1} . Aerobic respiration was 29 μ M min ^{? 1} , and sulfate reduction was 264 nmol cm ^{? 3} h ^{? 1} . During the dry season (precipitation 51 mm), cyanobacteria and anoxyphototrophs were less diverse and abundant, and T-RFLP patterns were less complex. The O ₂ production rate was reduced to 9 μ M min ^{? 1} , as was O ₂ consumption (7 μ M min ^{? 1} ) and sulfate reduction (26 nmol cm ^{? 3} h ^{? 1} ). Aragonite, calcite, halite, and quartz were the predominant minerals. Seasonal differences were found in the green and pink layers for both halite and quartz. Gypsum was not observed, likely due to a sample handling artifact. The fluctuations in community composition and metabolic activity, principally reflected in fluctuations in binding and trapping potential of the uppermost mat community, might be responsible for the observed differences in mineralogy.     

Cell Envelope of an Iron-Oxidizing Bacterium: Studies of Lipopolysaccharide and Peptidoglycan

Further structural detail is presented of the cell envelope of the chemolithotroph Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans). Thin sections of purified lipopolysaccharide (LPS) and peptidoglycan show structures comparable to those seen in the envelope of intact cells, whereas negative stains of LPS appear as sheets, or ribbons, or both. The sugars common to LPS, namely, heptose, glucose, galactose, mannose, and 2-keto-3-deoxyoctulosonate, were identified. The cations, iron, calcium, and magnesium, were associated with LPS. The purified LPS had a density of 1.28 and an uncorrected sedimentation coefficient of 99.9S.     

Degradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D) by a Hypersaline Microbial Mat and Related Functional Changes in the Mat Community

Microbial mats possibly possess degradation capacities for haloorganic pollutants because of their wide range of different functional groups of microorganisms combined with extreme diurnal changes in pH, oxygen, and sulfide gradients. In this study, 20 mg/l of the chlorinated herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to a pristine hypersaline cyanobacterial mat from Guerrero Negro, Mexico, under a light regime of 12 h dark/12 h light (600 mol photons/m²s). The loss of 2,4-D was followed by chemical GC analysis; functional changes within the mat were determined with microelectrodes for oxygen, photosynthesis, pH, and sulfide. The depletion of 2,4-D due to photooxidation or sorption processes was checked in control experiments. Within 13 days, the light/dark incubated mats degraded 97% of the herbicide, while in permanent darkness only 35% were degraded. Adsorption of 2,4-D to the mat material, agar, or glass walls was negligible (4.6%), whereas 21% of the herbicide was degraded photochemically. The 2,4-D removal rate in the light/dark incubations was comparable to values reported for soils. The phototrophic community of the mat was permanently inhibited by the 2,4-D addition by 17% on average. The sulfate reduction in the entire mat and the respiration in the photic zone were inhibited more strongly but returned to original levels. Since at the end of the experiment the photosynthetic and respiratory activity of the mats were almost as high as in the beginning and 2,4-D almost completely disappeared, we conclude that the examined mats represent a robust and effective system for the degradation of the herbicide where probably the aerobic heterotrophic population is a major player in the degradation process.This revised version was published online in November 2004 with corrections to Volume 48.     

In Vitro Study of Lipid Biosynthesis in an Anaerobically Methane-Oxidizing Microbial Mat

The anaerobic oxidation of methane (AOM) is a key process in the global methane cycle, and the majority of methane formed in marine sediments is oxidized in this way. Here we present results of an in vitro ¹³CH₄ labeling study (δ¹³CH₄, ~5,400‰) in which microorganisms that perform AOM in a microbial mat from the Black Sea were used. During 316 days of incubation, the ¹³C uptake into the mat biomass increased steadily, and there were remarkable differences for individual bacterial and archaeal lipid compounds. The greatest shifts were observed for bacterial fatty acids (e.g., hexadec-11-enoic acid [16:1Δ11]; difference between the δ¹³C at the start and the end of the experiment [Δδ¹³C_start-end], ~160‰). In contrast, bacterial glycerol diethers exhibited only slight changes in δ¹³C (Δδ¹³C_start-end, ~10‰). Differences were also found for individual archaeal lipids. Relatively high uptake of methane-derived carbon was observed for archaeol (Δδ¹³C_start-end, ~25‰), a monounsaturated archaeol, and biphytanes, whereas for sn-2-hydroxyarchaeol there was considerably less change in the δ¹³C (Δδ¹³C_start-end, ~2‰). Moreover, an increase in the uptake of ¹³C for compounds with a higher number of double bonds within a suite of polyunsaturated 2,6,10,15,19-pentamethyleicosenes indicated that in methanotrophic archaea there is a biosynthetic pathway similar to that proposed for methanogenic archaea. The presence of group-specific biomarkers (for ANME-1 and ANME-2 associations) and the observation that there were differences in ¹³C uptake into specific lipid compounds confirmed that multiple phylogenetically distinct microorganisms participate to various extents in biomass formation linked to AOM. However, the greater ¹³C uptake into the lipids of the sulfate-reducing bacteria (SRB) than into the lipids of archaea supports the hypothesis that there is autotrophic growth of SRB on small methane-derived carbon compounds supplied by the methane oxidizers.     

Germ Warfare in a Microbial Mat Community: CRISPRs Provide Insights into the Co-Evolution of Host and Viral Genomes

CRISPR arrays and associated cas genes are widespread in bacteria and archaea and confer acquired resistance to viruses. To examine viral immunity in the context of naturally evolving microbial populations we analyzed genomic data from two thermophilic Synechococcus isolates (Syn OS-A and Syn OS-B′) as well as a prokaryotic metagenome and viral metagenome derived from microbial mats in hotsprings at Yellowstone National Park. Two distinct CRISPR types, distinguished by the repeat sequence, are found in both the Syn OS-A and Syn OS-B′ genomes. The genome of Syn OS-A contains a third CRISPR type with a distinct repeat sequence, which is not found in Syn OS-B′, but appears to be shared with other microorganisms that inhabit the mat. The CRISPR repeats identified in the microbial metagenome are highly conserved, while the spacer sequences (hereafter referred to as “viritopes” to emphasize their critical role in viral immunity) were mostly unique and had no high identity matches when searched against GenBank. Searching the viritopes against the viral metagenome, however, yielded several matches with high similarity some of which were within a gene identified as a likely viral lysozyme/lysin protein. Analysis of viral metagenome sequences corresponding to this lysozyme/lysin protein revealed several mutations all of which translate into silent or conservative mutations which are unlikely to affect protein function, but may help the virus evade the host CRISPR resistance mechanism. These results demonstrate the varied challenges presented by a natural virus population, and support the notion that the CRISPR/viritope system must be able to adapt quickly to provide host immunity. The ability of metagenomics to track population-level variation in viritope sequences allows for a culture-independent method for evaluating the fast co-evolution of host and viral genomes and its consequence on the structuring of complex microbial communities.     

Microbial Life in a Fjord: Metagenomic Analysis of a Microbial Mat in Chilean Patagonia

The current study describes the taxonomic and functional composition of metagenomic sequences obtained from a filamentous microbial mat isolated from the Comau fjord, located in the northernmost part of the Chilean Patagonia. The taxonomic composition of the microbial community showed a high proportion of members of the Gammaproteobacteria, including a high number of sequences that were recruited to the genomes of Moritella marina MP-1 and Colwellia psycherythraea 34H, suggesting the presence of populations related to these two psychrophilic bacterial species. Functional analysis of the community indicated a high proportion of genes coding for the transport and metabolism of amino acids, as well as in energy production. Among the energy production functions, we found protein-coding genes for sulfate and nitrate reduction, both processes associated with Gammaproteobacteria-related sequences. This report provides the first examination of the taxonomic composition and genetic diversity associated with these conspicuous microbial mat communities and provides a framework for future microbial studies in the Comau fjord.     

Microbial Community Succession in an Unvegetated,Recently Deglaciated Soil

Primary succession is a fundamental process in macroecosystems; however, if and how soil development influences microbial community structure is poorly understood. Thus, we investigated changes in the bacterial community along a chronosequence of three unvegetated, early successional soils (∼20-year age gradient) from a receding glacier in southeastern Peru using molecular phylogenetic techniques. We found that evenness, phylogenetic diversity, and the number of phylotypes were lowest in the youngest soils, increased in the intermediate aged soils, and plateaued in the oldest soils. This increase in diversity was commensurate with an increase in the number of sequences related to common soil bacteria in the older soils, including members of the divisions Acidobacteria, Bacteroidetes, and Verrucomicrobia. Sequences related to the Comamonadaceae clade of the Betaproteobacteria were dominant in the youngest soil, decreased in abundance in the intermediate age soil, and were not detected in the oldest soil. These sequences are closely related to culturable heterotrophs from rock and ice environments, suggesting that they originated from organisms living within or below the glacier. Sequences related to a variety of nitrogen (N)-fixing clades within the Cyanobacteria were abundant along the chronosequence, comprising 6–40% of phylotypes along the age gradient. Although there was no obvious change in the overall abundance of cyanobacterial sequences along the chronosequence, there was a dramatic shift in the abundance of specific cyanobacterial phylotypes, with the intermediate aged soils containing the greatest diversity of these sequences. Most soil biogeochemical characteristics showed little change along this ∼20-year soil age gradient; however, soil N pools significantly increased with soil age, perhaps as a result of the activity of the N-fixing Cyanobacteria. Our results suggest that, like macrobial communities, soil microbial communities are structured by substrate age, and that they, too, undergo predictable changes through time.     

New Insight into Microbial Iron Oxidation as Revealed by the Proteomic Profile of an Obligate Iron-Oxidizing Chemolithoautotroph

Microaerophilic, neutrophilic, iron-oxidizing bacteria (FeOB) grow via the oxidation of reduced Fe(II) at or near neutral pH, in the presence of oxygen, making them relevant in numerous environments with elevated Fe(II) concentrations. However, the biochemical mechanisms for Fe(II) oxidation by these neutrophilic FeOB are unknown, and genetic markers for this process are unavailable. In the ocean, microaerophilic microorganisms in the genus Mariprofundus of the class Zetaproteobacteria are the only organisms known to chemolithoautotrophically oxidize Fe and concurrently biomineralize it in the form of twisted stalks of iron oxyhydroxides. The aim of this study was to identify highly expressed proteins associated with the electron transport chain of microaerophilic, neutrophilic FeOB. To this end, Mariprofundus ferrooxydans PV-1 was cultivated, and its proteins were extracted, assayed for redox activity, and analyzed via liquid chromatography-tandem mass spectrometry for identification of peptides. The results indicate that a cytochrome c₄, cbb₃-type cytochrome oxidase subunits, and an outer membrane cytochrome c were among the most highly expressed proteins and suggest an involvement in the process of aerobic, neutrophilic bacterial Fe oxidation. Proteins associated with alternative complex III, phosphate transport, carbon fixation, and biofilm formation were abundant, consistent with the lifestyle of Mariprofundus.     

Diel Migrations of Microorganisms within a Benthic, Hypersaline Mat Community

We studied the diel migrations of several species of microorganisms in a hypersaline, layered microbial mat. The migrations were quantified by repeated coring of the mat with glass capillary tubes. The resulting minicores were microscopically analyzed by using bright-field and epifluorescence (visible and infrared) microscopy to determine depths of coherent layers and were later dissected to determine direct microscopic counts of microorganisms. Microelectrode measurements of oxygen concentration, fiber optic microprobe measurements of light penetration within the mat, and incident irradiance measurements accompanied the minicore sampling. In addition, pigment content, photosynthesis and irradiance responses, the capacity for anoxygenic photosynthesis, and gliding speeds were determined for the migrating cyanobacteria. Heavily pigmented Oscillatoria sp. and Spirulina cf. subsalsa migrated downward into the mat during the early morning and remained deep until dusk, when upward migration occurred. The mean depth of the migration (not more than 0.4 to 0.5 mm) was directly correlated with the incident irradiance over the mat surface. We estimated that light intensity at the upper boundary of the migrating cyanobacteria was attenuated to such an extent that photoinhibition was effectively avoided but that intensities which saturated photosynthesis were maintained through most of the daylight hours. Light was a cue of paramount importance in triggering and modulating the migration of the cyanobacteria, even though the migrating phenomenon could not be explained solely in terms of a light response. We failed to detect diel migration patterns for other cyanobacterial species and filamentous anoxyphotobacteria. The sulfide-oxidizing bacterium Beggiatoa sp. migrated as a band that followed low oxygen concentrations within the mat during daylight hours. During the nighttime, part of this population migrated toward the mat surface, but a significant proportion remained deep.     

Sulfate-reducing bacteria in marine sediment (Aarhus Bay, Denmark): abundance and diversity related to geochemical zonation

In order to better understand the main factors that influence the distribution of sulfate-reducing bacteria (SRB), their population size and their metabolic activity in high- and low-sulfate zones, we studied the SRB diversity in 3- to 5-m-deep sediment cores, which comprised the entire sulfate reduction zone and the upper methanogenic zone. By combining EMA (ethidium monoazide that can only enter damaged/dead cells and may also bind to free DNA) treatment with real-time PCR, we determined the distributions of total intact bacteria (16S rDNA genes) and intact SRB ( dsrAB gene), their relative population sizes, and the proportion of dead cells or free DNA with depth. The abundance of SRB corresponded in average to 13% of the total bacterial community in the sulfate zone, 22% in the sulfate–methane transition zone and 8% in the methane zone. Compared with the total bacterial community, there were relatively less dead/damaged cells and free DNA present than among the SRB and this fraction did not change systematically with depth. By DGGE analysis, based on the amplification of the dsrA gene (400 bp), we found that the richness of SRB did not change with depth through the geochemical zones; but the clustering was related to the chemical zonation. A full-length clone library of the dsrAB gene (1900 bp) was constructed from four different depths (20, 110, 280 and 500 cm), and showed that the dsrAB genes in the near-surface sediment (20 cm) was mainly composed of sequences close to the Desulfobacteraceae , including marine complete and incomplete oxidizers such as Desulfosarcina , Desulfobacterium and Desulfococcus . The three other libraries were predominantly composed of Gram-positive SRB.     

Metagenomic Sequencing of an In Vitro-Simulated Microbial Community

Background

Microbial life dominates the earth, but many species are difficult or even impossible to study under laboratory conditions. Sequencing DNA directly from the environment, a technique commonly referred to as metagenomics, is an important tool for cataloging microbial life. This culture-independent approach involves collecting samples that include microbes in them, extracting DNA from the samples, and sequencing the DNA. A sample may contain many different microorganisms, macroorganisms, and even free-floating environmental DNA. A fundamental challenge in metagenomics has been estimating the abundance of organisms in a sample based on the frequency with which the organism''s DNA was observed in reads generated via DNA sequencing.

Methodology/Principal Findings

We created mixtures of ten microbial species for which genome sequences are known. Each mixture contained an equal number of cells of each species. We then extracted DNA from the mixtures, sequenced the DNA, and measured the frequency with which genomic regions from each organism was observed in the sequenced DNA. We found that the observed frequency of reads mapping to each organism did not reflect the equal numbers of cells that were known to be included in each mixture. The relative organism abundances varied significantly depending on the DNA extraction and sequencing protocol utilized.

Conclusions/Significance

We describe a new data resource for measuring the accuracy of metagenomic binning methods, created by in vitro-simulation of a metagenomic community. Our in vitro simulation can be used to complement previous in silico benchmark studies. In constructing a synthetic community and sequencing its metagenome, we encountered several sources of observation bias that likely affect most metagenomic experiments to date and present challenges for comparative metagenomic studies. DNA preparation methods have a particularly profound effect in our study, implying that samples prepared with different protocols are not suitable for comparative metagenomics.