Diatoms in Acid Mine Drainage and Their Role in the Formation of Iron-Rich Stromatolites

Adverse conditions in the acid mine drainage (AMD) system at the Green Valley mine, Indiana, limit diatom diversity to one species, Nitzschia tubicola. It is present in three distinct microbial consortia: Euglena mutabilis-dominated biofilm, diatomdominated biofilm, and diatom-exclusive biofilm. E. mutabilis dominates the most extensive biofilm, with lesser numbers of N. tubicola, other eukaryotes, and bacteria. Diatom-dominated biofilm occurs as isolated patches containing N. tubicola with minor fungal hyphae, filamentous algae, E. mutabilis, and bacteria. Diatom-exclusive biofilm is rare, composed entirely of N. tubicola.

Diatom distribution is influenced by seasonal and intraseasonal changes in water temperature and chemistry. Diatoms are absent in winter due to cool water temperatures. In summer, isolated patchy communities are present due to warmer water temperatures. In 2001, the diatom community expanded its distribution following a major rainfall that temporarily diluted the effluent, creating hospitable conditions for diatom growth. After several weeks when effluent returned to preexisting conditions, the diatom biofilm retreated to isolated patches, and E. mutabilis biofilm flourished.

Iron-rich stromatolites underlie the biofilms and consist of distinct laminae, recording spatial and temporal oscillations in physicochemical conditions and microbial activity. The stromatolites are composed of thin, wavy laminae with partially decayed E. mutabilis biofilm, representing microbial activity and iron precipitation under normal AMD conditions. Alternating with the wavy layers are thicker, porous, spongelike laminae composed of iron precipitated on and incorporated into radiating colonies of diatoms. These layers indicate episodic changes in water chemistry, allowing diatoms to temporarily dominate the system.     

Composition and Structure of Microbial Communities from Stromatolites of Hamelin Pool in Shark Bay, Western Australia

Stromatolites, organosedimentary structures formed by microbial activity, are found throughout the geological record and are important markers of biological history. More conspicuous in the past, stromatolites occur today in a few shallow marine environments, including Hamelin Pool in Shark Bay, Western Australia. Hamelin Pool stromatolites often have been considered contemporary analogs to ancient stromatolites, yet little is known about the microbial communities that build them. We used DNA-based molecular phylogenetic methods that do not require cultivation to study the microbial diversity of an irregular stromatolite and of the surface and interior of a domal stromatolite. To identify the constituents of the stromatolite communities, small subunit rRNA genes were amplified by PCR from community genomic DNA with universal primers, cloned, sequenced, and compared to known rRNA genes. The communities were highly diverse and novel. The average sequence identity of Hamelin Pool sequences compared to the >200,000 known rRNA sequences was only ~92%. Clone libraries were ~90% bacterial and ~10% archaeal, and eucaryotic rRNA genes were not detected in the libraries. The most abundant sequences were representative of novel proteobacteria (~28%), planctomycetes (~17%), and actinobacteria (~14%). Sequences representative of cyanobacteria, long considered to dominate these communities, comprised <5% of clones. Approximately 10% of the sequences were most closely related to those of α-proteobacterial anoxygenic phototrophs. These results provide a framework for understanding the kinds of organisms that build contemporary stromatolites, their ecology, and their relevance to stromatolites preserved in the geological record.     

Multi-trichomous cyanobacterial microfossils from the Mesoproterozoic Gaoyuzhuang Formation, China: Paleoecological and taxonomic implications

Populations of the multi-trichomous microbial fossil Eoschizothrix composita n.gen. et sp. are preserved in growth position in silicified stratiform stromatolites of the Gaoyuzhuang Formation, Hebei Province, northern China. The microbial fossils consist predominantly of preserved sheaths, although several specimens retain shriveled remains of trichomes within sheaths. Comparisons with modern morphological counterparts, including shape, growth habit and orientation, degradational sequences, and habitat, support the interpretation of the multi-trichomous microfossils as cyanobacteria, which acted as frame-builders of ancient stromatolites. The distribution and orientation of multi-trichomous microfossils within a synsedimentary context reveal their behavioral responses to sedimentation regime. Horizontally spread, interwoven mats formed during periods of sedimentary stasis. During periods of rapid sediment influx, the filaments assumed an upright orientation, possibly to avoid accumulating particles. This is the first record of fossil stromatolite-building multi-trichomous cyanobacterial which underscores early morphological and functional diversification in cyanobacterial evolution.     

Influence of microbial biofilms on the preservation of primary soft tissue in fossil and extant archosaurs

Background

Mineralized and permineralized bone is the most common form of fossilization in the vertebrate record. Preservation of gross soft tissues is extremely rare, but recent studies have suggested that primary soft tissues and biomolecules are more commonly preserved within preserved bones than had been presumed. Some of these claims have been challenged, with presentation of evidence suggesting that some of the structures are microbial artifacts, not primary soft tissues. The identification of biomolecules in fossil vertebrate extracts from a specimen of Brachylophosaurus canadensis has shown the interpretation of preserved organic remains as microbial biofilm to be highly unlikely. These discussions also propose a variety of potential mechanisms that would permit the preservation of soft-tissues in vertebrate fossils over geologic time.

Methodology/Principal Findings

This study experimentally examines the role of microbial biofilms in soft-tissue preservation in vertebrate fossils by quantitatively establishing the growth and morphology of biofilms on extant archosaur bone. These results are microscopically and morphologically compared with soft-tissue extracts from vertebrate fossils from the Hell Creek Formation of southeastern Montana (Latest Maastrichtian) in order to investigate the potential role of microbial biofilms on the preservation of fossil bone and bound organic matter in a variety of taphonomic settings. Based on these analyses, we highlight a mechanism whereby this bound organic matter may be preserved.

Conclusions/Significance

Results of the study indicate that the crystallization of microbial biofilms on decomposing organic matter within vertebrate bone in early taphonomic stages may contribute to the preservation of primary soft tissues deeper in the bone structure.     

Metagenomic and metaproteomic analyses of Accumulibacter phosphatis‐enriched floccular and granular biofilm

Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here we operated two laboratory‐scale sequencing batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal. Reactors formed two distinct biofilms, one floccular biofilm, consisting of small, loose, microbial aggregates, and one granular biofilm, forming larger, dense, spherical aggregates. Using metagenomic and metaproteomic methods, we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. To understand biofilm differences, we compared protein abundances that were statistically enriched in both biofilm states. Floccular biofilms were enriched with pathogenic secretion systems suggesting a highly competitive microbial community. Comparatively, granular biofilms revealed a high‐stress environment with evidence of nutrient starvation, phage predation pressure, and increased extracellular polymeric substance and cell lysis. Granular biofilms were enriched in outer membrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter‐enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.     

Interactions among endophytic bacteria and fungi: Effects and potentials

Plants benefit extensively by harbouring endophytic microbes. They promote plant growth and confer enhanced resistance to various pathogens. However, the way the interactions among endophytes influence the plant productivity has not been explained. Present study experimentally showed that endophytes isolated from rice (Oryza sativa) used as the test plant produced two types of interactions; biofilms (bacteria attached to mycelia) and mixed cultures with no such attachments. Acidity, as measured by pH in cultures with biofilms was higher than that of fungi alone, bacteria alone or the mixed cultures. Production of indoleacetic acid like substances (IAAS) of biofilms was higher than that of mixed cultures, fungi or bacteria. Bacteria and fungi produced higher quantities of IAAS than mixed cultures. In mixed cultures, the potential of IAAS production of resident microbes was reduced considerably. There was a negative relationship between IAAS and pH of the biofilms, indicating that IAAS was the main contributor to the acidity. However, such a relationship was not observed in mixed cultures. Microbial acid production is important for suppressing plant pathogens. Thus the biofilm formation in endophytic environment seems to be very important for healthy and improved plant growth. However, it is unlikely that an interaction among endophytes takes place naturally in the endophytic environment, due to physical barriers of plant tissues. Further, critical cell density dependant quorum sensing that leads to biofilm formation may not occur in the endophytic environment as there is a limited space. As suchin vitro production and application of beneficial biofilmed inocula of endophytes are important for improved plant production in any agro-ecosystem. The conventional practice of plant inoculation with monocultures or mixed cultures of effective microbes may not give the highest microbial effect, which may only be achieved by biofilm formation.     

Viscoelastic properties of Staphylococcus aureus and Staphylococcus epidermidis mono‐microbial biofilms

The viscoelastic properties of mono‐microbial biofilms produced by ocular and reference staphylococcal strains were investigated. The microorganisms were characterized for their haemolytic activity and agr typing and the biofilms, grown on stainless steel surface under static conditions, were analysed by Confocal Laser Scanning Microscopy. Static and dynamic rheometric tests were carried out to determine the steady‐flow viscosity and the elastic and viscous moduli. The analysed biofilms showed the typical time‐dependent behaviour of viscoelastic materials with considerable elasticity and mechanical stability except for Staphylococcus aureus ATCC 29213 biofilm which showed a very fragile structure. In particular, S. aureus 6ME biofilm was more compact than other staphylococcal biofilms studied with a yield stress ranging between 2 and 3 Pa. The data obtained in this work could represent a starting point for developing new therapeutic strategies against biofilm‐associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier.     

Bacterial Insights into the Formation of Opaline Stromatolites from the Chimalacatepec Lava Tube System,Mexico

Abstract

Cave lithifying systems are excellent models to study biomineralization in the dark. The Chimalacatepec Lava Tube System in Mexico harbors diverse biospeleothems where previous studies suggest that the formation of opaline terrestrial stromatolites is related to microorganisms in contiguous mats. However, there is no information regarding their characterization and their role in mineral formation. In this study, we characterized the bacterial and archaeal composition of microbial mats and stromatolites and suggested the main processes involved in the genesis of opaline stromatolites. Our results showed that the microbial mats and stromatolites have a similar 16S rRNA gene composition, but stromatolites contain more Actinobacteria, which have been previously found in other lava tubes together with other key bacteria. Microorganisms found here belonged to groups with the potential to fix carbon and degrade organic matter. We propose that the synergic interaction of autotrophic and heterotrophic microorganisms that thrive in the dark might be inducing carbonate precipitation within the Ca-enriched extracellular polymeric substances (EPS), generating opal-A and calcite laminae. The similar 16S rRNA gene fingerprint and the presence of potential pathways that induce carbonate precipitation in opaline stromatolites and microbial mats suggest that microbial mats lithify and contribute to the stromatolite biotic genesis.     

Acid microenvironments in microbial biofilms of antarctic endolithic microecosystems

Antarctic endolithic microecosystems harbour distinct biofilms. The lithic substrate and the microorganisms comprising these films are intimately linked, leading to complex mineral-microbe interactions. Hence, the microhabitats and microenvironments of these microecosystems are not only determined by the physicochemical features of the lithic substrate, but are also conditioned by the biological components of these biofilms. The Antarctic biofilms analysed in this study are characterized by the presence of extracellular polymer substances and acid microenvironments in the proximity of the cells; cyanobacteria appearing as key components. On ultrastructural analysis, these endolithic cyanobacteria showed differences in sheath organization, probably related to their spatial position in the lithic substrate. It is proposed that in this type of ecosystem, biofilm structure could favour the formation of microsites with specific physicochemical conditions appropriate for the survival of microbial communities in this extreme environment.     

Fossilization of Acidophilic Microorganisms

This study examines fossil microorganisms found in iron-rich deposits in an extreme acidic environment, the Tinto River in SW Spain. Both electron microscopy (SEM and TEM) and non-destructive in situ microanalytical techniques (EDS, EMP and XPS) were used to determine the role of permineralization and encrustation in preserving microorganisms forming biofilms in the sediments. Unicellular algae were preserved by silica permineralization of their cell walls. Bacterial biofilms were preserved as molds by epicellular deposition of schwertmannite around them. In the case of fungi and filamentous algae, we observed permineralization of cell structures by schwertmannite in the sediments. The extracellular polymeric matrix around the cells was also preserved through permineralization of the fibrillar component. The process of permineralization and deposition of iron-rich precipitates present in the acidic waters of Rio Tinto served to preserve many microfossils in an oxidizing environment, in which organic compounds would not normally be expected to persist. Studies of microbial fossil formation mechanisms in modern extreme environments should focus on defining criteria to identify inorganic traces of microbial life in past environments on Earth or other planets.     

Quantifying CaCO3 Microprecipitates Within Developing Surface Mats of Marine Stromatolites Using GIS and Digital Image Analysis

The unique geochemical coupling of organic molecules and mineral CaCO₃ provides a fluorescence signature detectable using conventional confocal scanning laser microscopy (CSLM). The surface microbial mats of open-water marine stromatolites (Bahamas) exist in a continuum of states ranging from a Type 1 (i.e., nonlithifying) to Type 2 (i.e., lithified micritic laminae present) to Type 3 (i.e., fused grain layer). An approach was developed here, that utilizes geographical information systems (GIS) and digital image analysis, coupled with CSLM to estimate concentrations of calcium carbonate precipitates in developing marine stromatolites. We propose that the area occupied by particles within each image can be used to estimate concentrations of precipitates. Fluorescent polymeric microbeads and bacteria were used to calibrate the approach. We used this approach to demonstrate that CaCO₃ precipitates in lithifying layers were quantifiable and significantly different (p < 0.0001) from those in nonlithifying layers. The approach provided a useful tool for the unambiguous assessment of relative changes in microbial precipitates occurring over small ( μ m to mm) spatial scales, and that characterize the formation of lithified layers (micritic laminae) in open-water marine stromatolites.     

Bioclaustration trace fossils in epeiric shallow marine stromatolites: the Cretaceous‐Palaeogene Yacoraite Formation,Northwestern Argentina

The shallow carbonate facies at the top of the Yacoraite Formation (Late Cretaceous–Early Palaeocene) in the Metán sub‐basin, Salta Basin (Cretaceous‐Eocene), northern Argentina, have domal stromatolitic boundstones with peculiar cavities, interpreted here as bioclaustrations. The cavities appear to have been produced by organisms that lived within the microbial mat contemporarily with its growth, producing a distinctive ichnofabric. This is the oldest reported record of bioclaustrations in stromatolites, and the first in shallow marine environments. The interpretation of the facies suggests a stressed shallow, restricted setting with variations in salinity, represented by an intertidal environment with an extensive tidal flat. Bioclaustrations, stromatolites, endobiont Yacoraite Formation (Cretaceous‐Palaeogene), Northwestern Argentina.     

Formation of nesquehonite and other minerals as a consequence of biofilm dehydration

A microbial biofilm community was established over 971 days within gravel in an aquarium so as to model biofouling of an aquifer. When the water was allowed to evaporate slowly, white crystalline deposits, containing several carbonate and sulphate minerals including nesquehonite (MgCO₃.3H2O), were seen at the highest points on the surface of the biofouled gravel. No such deposits occurred in regions lacking biofilms. These crystals appeared to originate from evaporation of dissolved salts which had migrated through the biofilm. Surfaceadherent microbial biofilms may conceivably provide a conduit for solute transport in porous media such as soils and aquifers.     

The Discovery of Stromatolites Developing at 3570 m above Sea Level in a High-Altitude Volcanic Lake Socompa,Argentinean Andes

We describe stromatolites forming at an altitude of 3570 m at the shore of a volcanic lake Socompa, Argentinean Andes. The water at the site of stromatolites formation is alkaline, hypersaline, rich in inorganic nutrients, very rich in arsenic, and warm (20–24°C) due to a hydrothermal input. The stromatolites do not lithify, but form broad, rounded and low-domed bioherms dominated by diatom frustules and aragonite micro-crystals agglutinated by extracellular substances. In comparison to other modern stromatolites, they harbour an atypical microbial community characterized by highly abundant representatives of Deinococcus-Thermus, Rhodobacteraceae, Desulfobacterales and Spirochaetes. Additionally, a high proportion of the sequences that could not be classified at phylum level showed less than 80% identity to the best hit in the NCBI database, suggesting the presence of novel distant lineages. The primary production in the stromatolites is generally high and likely dominated by Microcoleus sp. Through negative phototaxis, the location of these cyanobacteria in the stromatolites is controlled by UV light, which greatly influences their photosynthetic activity. Diatoms, dominated by Amphora sp., are abundant in the anoxic, sulfidic and essentially dark parts of the stromatolites. Although their origin in the stromatolites is unclear, they are possibly an important source of anaerobically degraded organic matter that induces in situ aragonite precipitation. To the best of our knowledge, this is so far the highest altitude with documented actively forming stromatolites. Their generally rich, diverse and to a large extent novel microbial community likely harbours valuable genetic and proteomic reserves, and thus deserves active protection. Furthermore, since the stromatolites flourish in an environment characterized by a multitude of extremes, including high exposure to UV radiation, they can be an excellent model system for studying microbial adaptations under conditions that, at least in part, resemble those during the early phase of life evolution on Earth.     

Metal immobilisation by biofilms: Mechanisms and analytical tools

In biofilm environments, heavy metal and radionuclide pollutants are removed by a variety of mechanisms, including biosorption, precipitation as sulfides or phosphates and microbial reductive precipitation. Even if the elemental composition and localization of the precipitate trapped in the biofilm is well described thanks to spectroscopic and microscopic techniques, this review highlights that little is known about metal immobilisation mechanisms in microbial biofilms, i.e., mass transfer of metals, mechanisms involved in (bio)sorption and precipitation and the influence of physicochemical micro-environments within the biofilm matrix. The review shows the advantage of using a combination of different techniques to evaluate the fate of metals within microbial biofilms. By combining a variety of techniques (e.g., selective extraction, microscopy, spectroscopy and miniaturised sensors ...), it is possible to gain high-resolution structural and chemical information of biofilms on a level of the individual cell. This approach will facilitate the characterization of the metal immobilisation sites and the metal sorption and (bio)crystallisation mechanisms in biofilms. The results provided by the combination of these techniques will allow to predict the amount of metal accumulation in biofilms as well as their chemical speciation. This review demonstrates that an interdisciplinary approach is required to study metal fate within the biofilm matrix. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phototrophic marine benthic microbiomes: the ecophysiology of these biological entities

Phototrophic biofilms are multispecies, self-sustaining and largely closed microbial ecosystems. They form macroscopic structures such as microbial mats and stromatolites. These sunlight-driven consortia consist of a number of functional groups of microorganisms that recycle the elements internally. Particularly, the sulfur cycle is discussed in more detail as this is fundamental to marine benthic microbial communities and because recently exciting new insights have been obtained. The cycling of elements demands a tight tuning of the various metabolic processes and require cooperation between the different groups of microorganisms. This is likely achieved through cell-to-cell communication and a biological clock. Biofilms may be considered as a macroscopic biological entity with its own physiology. We review the various components of some marine phototrophic biofilms and discuss their roles in the system. The importance of extracellular polymeric substances (EPS) as the matrix for biofilm metabolism and as substrate for biofilm microorganisms is discussed. We particularly assess the importance of extracellular DNA, horizontal gene transfer and viruses for the generation of genetic diversity and innovation, and for rendering resilience to external forcing to these biological entities.     

Modeling bioaffinity-based targeted delivery of antimicrobials to Escherichia coli biofilms using yeast microparticles. Part II: Parameter evaluation and validation

The design of bioaffinity-based targeted delivery systems for biofilm inactivation may require a comprehensive understanding of physicochemical and biochemical properties of biobased antimicrobial particles and their interactions with biofilm. In this study, Escherichia coli biofilm inactivation by chlorine-charged yeast microparticles was numerically simulated, and the roles of chemical stability, binding affinity, and controlled release of this targeted delivery system were assessed using this numerical simulation. The simulation results were experimentally validated using two different types of yeast microparticles. The results of this study illustrate that chorine stability achieved by yeast microparticles was a key factor for improved biofilm inactivation in an organic-rich environment (>6 additional log reduction in 20 min compared to the free chlorine treatment). Moreover, the binding affinity of yeast microparticles to E. coli biofilms was another key factor for an enhanced inactivation of biofilm, as a 10-fold increase in binding rate resulted in a 4.2-fold faster inactivation. Overall, the mechanistic modeling framework developed in this study could guide the design and development of biobased particles for targeted inactivation of biofilms.     

Mechanisms of Bacillus cereus biofilm formation: an investigation of the physicochemical characteristics of cell surfaces and extracellular proteins

Microbial biofilms contribute to biofouling in a wide range of processes from medical implants to processed food. The extracellular polymeric substances (EPS) are implicated in imparting biofilms with structural stability and resistance to cleaning products. Still, very little is known about the structural role of the EPS in Gram-positive systems. Here, we have compared the cell surface and EPS of surface-attached (biofilm) and free-floating (planktonic) cells of Bacillus cereus, an organism routinely isolated from within biofilms on different surfaces. Our results indicate that the surface properties of cells change during biofilm formation and that the EPS proteins function as non-specific adhesions during biofilm formation. The physicochemical traits of the cell surface and the EPS proteins give us an insight into the forces that drive biofilm formation and maintenance in B. cereus.     

The impact of biofilm-forming potential and tafi production on transport of environmental <Emphasis Type="Italic">Salmonella</Emphasis> through unsaturated porous media

Understanding the factors influencing the transport of microbial pathogens, such as Salmonella and Escherichia coli, through porous media is critical to protecting drinking water supplies. The production of biofilms, along with individual biofilm-associated components, such as tafi, is believed to hinder transport of microorganisms through soil. This study investigated the relationship between biofilm formation and tafi production and the transport of environmental Salmonella through porous media. Thirty-two Salmonella isolates were initially assayed for their ability to form biofilms, from which a subset of these was selected to represent a range of high and low biofilm-formation potential and tafi formation capabilities. These were subsequently examined in unsaturated sand columns for transport characteristics. No obvious correlation was observed between Salmonella phenotypes and column retention. The results indicated that while transport of well-characterized laboratory E. coli strains can often be hindered by the presence of tafi and the potential to form biofilms, the presence of tafi did not retard the transport of the Salmonella strains.     

Biotic and Abiotic Imprints on Mg-Rich Stromatolites: Lessons from Lake Salda,SW Turkey

Abstract

Modern hydrated Mg rich stromatolites are actively growing along the shallow shorelines of Lake Salda (SW Turkey). An integrated approach involving isotopic, mineralogical, microscopic, and organic/geochemical techniques along with culture-independent molecular methods were applied to various lake samples to assess the role of microbial processes on stromatolite formation. This study further explores the biosignature preservation potential of fossil stromatolites by comparing with textures, lipid profiles and isotopic composition of the modern stromatolites. Similar lipid profile and δ¹³C isotope values in active and fossil stromatolites argue that CO₂ cycling delicately balanced between photosynthetic and heterotrophic (aerobic) activity as in the active ones may have regulated stromatolite formation in the lake. A decrease in the exopolymeric substances (EPS) profile of the mat and concurrent hydromagnesite precipitation imply a critical role for EPS in the formation of stromatolite. Consistently, a discrete, discontinuous lamination and clotted micropeloidal textures with cyanobacterial remnants in the fossil stromatolites likely refer to partial degradation of EPS, creating local nucleation sites and allowing precipitation of hydrated Mg minerals and provide a link to the active microbial mat in the modern stromatolites. Our results for the first time provide strong evidence for close coupling of cyanobacterial photosynthesis and aerobic heterotrophic respiration on hydromagnesite textures involved in the stromatolite formation of Lake Salda. The creation of photosynthesis induced high-pH conditions combined with a change in the amount and properties of the EPS and the repetition of these processes over time seems to be a possible pathway for stromatolite growth in the lake. Understanding these microbial symbioses and their mineralized records may provide new insights on the formation mechanism of Mg-rich carbonates not only for terrestrial geological records but also for planetary bodies like Mars, where hydrated Mg-carbonate deposits have been identified in possible paleolake deposits at Jezero crater, the landing site of the NASA Mars 2020 rover.