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

We constructed a small flow chamber in which suboxic medium containing 60 to 120 μM FeCl₂ flowed up through a sample well into an aerated reservoir, thereby creating an suboxic-oxic interface similar to the physicochemical conditions that exist in natural iron seeps. When microbial mat material from the Marselisborg iron seep that contained up to 10⁹ bacterial cells per cm³ (D. Emerson and N. P. Revsbech, Appl. Environ. Microbiol. 60:4022-4031, 1994) was placed in the sample well of the chamber, essentially all of the Fe²⁺ flowing through the sample well was oxidized at rates of up to 1,200 nmol of Fe²⁺ oxidized per h per cm³ of mat material. The oxidation rates of samples of the mat that were pasteurized prior to inoculation were only about 20 to 50% of the oxidation rates of unpasteurized samples. Sodium azide also significantly inhibited oxidation. These results suggest that at least 50% and up to 80% of the Fe oxidation in the chamber were actively mediated by the microbes in the mat. It also appeared that Fe stimulated the growth of the community since chambers fed with FeCl₂ accumulated masses of either filamentous or particulate growth, both in the sample well and attached to the walls of the chamber. Control chambers that did not receive FeCl₂ showed no sign of such growth. Furthermore, after 4 to 5 days the chambers fed with FeCl₂ contained 35 to 75% more protein than chambers not supplemented with FeCl₂. Leptothrix ochracea and, to a lesser extent, Gallionella spp. were responsible for the filamentous growth, and the sheaths and stalks, respectively, of these two organisms harbored large numbers of Fe-encrusted, nonappendaged unicellular bacteria. In chambers where particulate growth predominated, the unicellular bacteria alone appeared to be the primary agents of iron oxidation. These results provide the first clear evidence that the “iron bacteria” commonly found associated with neutral-pH iron seeps are responsible for most of the iron oxidation and that the presence of ferrous iron appears to stimulate the growth of these organisms.     

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.     

Identification and Distribution of High-Abundance Proteins in the Octopus Spring Microbial Mat Community

A shotgun metaproteomics approach was employed to identify proteins in a hot spring microbial mat community. We identified 202 proteins encompassing 19 known functions from 12 known phyla. Importantly, we identified two key enzymes involved in the 3-hydroxypropionate CO₂ fixation pathway in uncultivated Roseiflexus spp., which are known photoheterotrophs.     

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.     

Microelectrode Studies of Interstitial Water Chemistry and Photosynthetic Activity in a Hot Spring Microbial Mat

Microelectrodes were used to measure oxygen, pH, and oxygenic photosynthetic activity in a hot spring microbial mat (Octopus Spring, Yellowstone National Park), where the cyanobacterium Synechococcus lividus and the filamentous bacterium Chloroflexus aurantiacus are the only known phototrophs. The data showed very high biological activities in the topmost layers of the microbial mat, resulting in extreme values for oxygen and pH. At a 1-mm depth at a 55°C site, oxygen and pH reached 900 μM and 9.4, respectively, just after solar noon, whereas anoxic conditions with a pH of 7.2 were measured before sunrise. Although diurnal changes between these extremes occurred over hours during a diurnal cycle, microbial activity was great enough to give the same response in 1 to 2 min after artificial shading. Oxygenic photosynthesis was confined to a 0.5- to 1.1-mm layer at sites with temperatures at or above about 50°C, with maximum activities in the 55 to 60°C region. The data suggest that S. lividus is the dominant primary producer of the mat.     

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.     

Maternal Vitamin D Status and Offspring Bone Fractures: Prospective Study over Two Decades in Aarhus City,Denmark

Background

Studies investigating the association between maternal vitamin D status and offspring bone mass measured by dual-energy X-ray absorptiometry (DXA) during childhood have shown conflicting results.

Purpose

We used occurrence of bone fractures up to the age of 18 as a measure reflecting offspring bone mass and related that to maternal vitamin D status.

Methods

The Danish Fetal Origins 1988 Cohort recruited 965 pregnant women during 1988–89 at their 30^th gestation week antenatal midwife visit. A blood sample was drawn and serum was stored, which later was analyzed for the concentration of 25-hydroxyvitamin D (25(OH)D) by the liquid chromatography coupled with a tandem mass spectrometric method (LC-MS/MS). Outcome was diagnosis of first time bone fractures extracted from the Danish National Patient Register.

Results

Vitamin D status was available for 850 women. The median (5^th–95^th percentile) 25(OH)D was 76.2 (23.0–152.1) nmol/l. During follow up 294 children were registered with at least one bone fracture diagnosis. Multivariable Cox regression models using age as the underlying time scale indicated no overall association between maternal vitamin D status and first time bone fractures. However, there was a significantly increased hazard ratio (HR) during childhood for those who had maternal blood drawn in Dec/Jan/Feb compared with Jun/Jul/Aug (HR: 1.75, 95%CI: 1.11–2.74). Adjustment for vitamin D status strengthened this association (1.82, 1.12–2.97), which indicated a potential seasonal impact on offspring fractures independent of maternal vitamin D status. In a sensitivity analysis we found a borderline significant inverse association between continuous concentrations of 25(OH)D and offspring forearm fractures (P = 0.054).

Conclusion

Overall, our results did not substantiate an association between maternal vitamin D status and offspring bone fractures. Further studies on this subject are needed, but the study populations must be large enough to allow for subdivision of fractures.