Polyphasic detection of cyanobacteria in terrestrial biofilms

Cyanobacterial populations detected on buildings by traditional methods are mainly filamentous, whereas direct microscopy shows that they are principally coccoid morphotypes that often cannot be isolated in culture, but may grow on artificial media when the spatial biofilm relationships are maintained. The polyphasic strategy described here was to select morphologically distinct colonies from rehydrated biofilms for direct DNA amplification, allowing uncultured organisms to be sequenced and their morphology to be characterized by microscopy. DNA data banks currently contain many entries for cyanobacteria of unrecorded morphology, which does not facilitate identification, although genetic variability in a population may be assessed. The sequence homologies of the present biofilm organisms (EMBL accession numbers AJ619681 to 619690) with those in DNA databanks were low, indicating differences between xerophytic cyanobacteria on walls and aquatic species comprising the majority in the databases. Further development of databases for the populations found in this environment, subject to temperature extremes, repeated desiccation and high UV and salt levels, is required.     

Microbial stabilization of sediments in a recent Salina, Lake Aghormi, Siwa Oasis, Egypt

Stabilization of sediments by microbial mats and biofilms were studied in detail in Lake Aghormi, Siwa Oasis, Egypt. The study has shown that microbial mat assemblages, particularly filamentous cyanobacteria, with their extracellular polymeric substances (EPS) are capable of effectively stabilizing sediments. The microbial mats in the siliciclastic environments of Lake Aghormi display distinctive sedimentary structures (microbially induced sedimentary structures), including multidirected ripple marks, microbial patches, petee structures, erosional remnants and pockets, and gas domes. Scanning electron microscopy study of the sediment surface colonized by cyanobacteria revealed that filamentous types are the most effective stabilizing organisms. Filamentous cyanobacteria and their EPS construct a network, interweave depositional grains of the sediment surface, envelope the particles, and glue them together. The studied biofilm is so thick forming a spider-web structure that totally coat the particles in such a way the morphology of the particles is masked.     

Ultrastructural and genetic characteristics of endolithic cyanobacterial biofilms colonizing Antarctic granite rocks

The precise identification of the cyanobacteria that comprise an endolithic biofilm is hindered by difficulties in culturing the organisms found in these biofilms and a lack of previous molecular and ultrastructural data. This study characterizes, both at the ultrastructural and molecular level, two different cyanobacterial biofilms found in fissures of granite from continental Antarctica. Electron microscopy revealed structural differences between the two biofilms. One was only loosely adhered to the substrate, while the other biofilm showed a closer association between cells and rock minerals and was tightly attached to the substrate. Cells from both biofilms where ultrastructurally distinct, displaying, for instance, clear differences in their sheaths. The amounts of EPS and their organization associated with the cyanobacteria may determine the differences in adhesion and effects on the lithic substrate observed in the biofilms. By sequencing part of the 16S rRNA gene, the two cyanobacteria were also genetically characterized. The gene sequence of the cells comprising the biofilm that was tightly attached to the lithic substrate showed most homology with that of an endolithic cyanobacterium from Switzerland (AY153458), and the cyanobacterial type loosely adhered to the rock, clustered with Acaryochloris marina, the only organism unequivocally known to contain chlorophyll d. This study reveals the presence of at least two different types of endolithic biofilm, dominated each by a single type of cyanobacterium, able to withstand the harsh conditions of the Antarctic climate.     

Microbial diversity of biofilms in dental unit water systems

We investigated the microbial diversity of biofilms found in dental unit water systems (DUWS) by three methods. The first was microscopic examination by scanning electron microscopy (SEM), acridine orange staining, and fluorescent in situ hybridization (FISH). Most bacteria present in the biofilm were viable. FISH detected the beta and gamma, but not the alpha, subclasses of Proteobacteria: In the second method, 55 cultivated biofilm isolates were identified with the Biolog system, fatty acid analysis, and 16S ribosomal DNA (rDNA) sequencing. Only 16S identified all 55 isolates, which represented 13 genera. The most common organisms, as shown by analyses of 16S rDNA, belonged to the genera Afipia (28%) and Sphingomonas (16%). The third method was a culture-independent direct amplification and sequencing of 165 subclones from community biofilm 16S rDNA. This method revealed 40 genera: the most common ones included Leptospira (20%), Sphingomonas (14%), Bacillus (7%), Escherichia (6%), Geobacter (5%), and Pseudomonas (5%). Some of these organisms may be opportunistic pathogens. Our results have demonstrated that a biofilm in a health care setting may harbor a vast diversity of organisms. The results also reflect the limitations of culture-based techniques to detect and identify bacteria. Although this is the greatest diversity reported in DUWS biofilms, other genera may have been missed. Using a technique based on jackknife subsampling, we projected that a 25-fold increase in the number of subclones sequenced would approximately double the number of genera observed, reflecting the richness and high diversity of microbial communities in these biofilms.     

Unraveling Cyanobacteria Ecology in Wastewater Treatment Plants (WWTP)

Cyanobacteria may be important components of wastewater treatment plants’ (WWTP) biological treatment, reaching levels of 100% of the total phytoplankton density in some systems. The occurrence of cyanobacteria and their associated toxins in these systems present a risk to the aquatic environments and to public health, changing drastically the ecology of microbial communities and associated organisms. Many studies reveal that cyanotoxins, namely microcystins may not act as antibacterial compounds but they might have negative impacts on protozoans, inhibiting their growing and respiration rates and leading to changes in cellular morphology, decreasing consequently the treatment efficacy in WWTP. On the other side, flagellates and ciliates may ingest some cyanobacteria species while the formation of colonies by these prokaryotes may be seen as a defense mechanism against predation. Problems regarding the occurrence of cyanobacteria in WWTP are not limited to toxin production. Other cyanobacterial secondary metabolites may act as antibacterial compounds leading to the disruption of bacterial communities that biologically convert organic materials in WWTP being fundamental to the efficacy of the process. Studies reveal that the potential antibacterial capacity differs according to cyanobacteria specie and it seems to be more effective in Gram (+) bacteria. Thus, to understand the effects of cyanobacterial communities in the efficiency of the waste water treatment it will be necessary to unravel the complex interactions between cyanobacterial populations, bacteria, and protozoa in WWTP in situ studies.     

Allelopathic control of cyanobacterial blooms by periphyton biofilms

Periphyton biofilms are natural mixtures comprised of photoautotrophic and heterotrophic complex microorganisms. In this work, the inhibition effects of periphyton biofilms on cyanobacterial blooms were studied in pilot and field trials. Results show that the cyanobacterial species responsible for the blooms had an upper nutrient concentration threshold, below which it could not effectively compete with other organisms in the periphyton. The disappearance of the cyanobacterial blooms was due to the allelopathy between the cyanobacteria and periphyton biofilm. In particular, it was found that the periphyton biofilm could produce water-soluble allelochemicals such as indole and 3-oxo-α-ionone to significantly inhibit the growth of the cyanobacteria. These allelochemicals are able to damage the thylakoid membranes of the cyanobacteria, interrupt the electron transport in photosystem II, decrease effective quantum yields, and eventually lead to the failure of photosynthesis. A comprehensive discussion on the ecological consequences of these findings is also presented. This work demonstrates the potential of periphyton biofilm to be used as an environmentally friendly ecological engineering solution for (i) the control of cyanobacterial blooms and (ii) a transitional means for the construction of beneficial conditions for ecosystem restoration. In addition, this work provides significant insights into the competitive relationships between algae and biofilms.     

Microbial Biofilms on the Sandstone Monuments of the Angkor Wat Complex,Cambodia

Discoloring biofilms from Cambodian temples Angkor Wat, Preah Khan, and the Bayon and West Prasat in Angkor Thom contained a microbial community dominated by coccoid cyanobacteria. Molecular analysis identified Chroococcidiopsis as major colonizer, but low similarity values (<95%) suggested a similar genus or species not present in the databases. In only two of the six sites sampled were filamentous cyanobacteria, Microcoleus, Leptolyngbya, and Scytonema, found; the first two detected by sequencing of 16S rRNA gene library clones from samples of a moist green biofilm on internal walls in Preah Khan, where Lyngbya (possibly synonymous with Microcoleus) was seen by direct microscopy as major colonizer. Scytonema was detected also by microscopy on an internal wall in the Bayon. This suggests that filamentous cyanobacteria are more prevalent in internal (high moisture) areas. Heterotrophic bacteria were found in all samples. DNA sequencing of bands from DGGE gels identified Proteobacteria (Stenotrophomonas maltophilia and Methylobacterium radiotolerans) and Firmicutes (Bacillus sp., Bacillus niacini, Bacillus sporothermodurans, Lysinibacillus fusiformis, Paenibacillus sp., Paenibacillus panacisoli, and Paenibacillus zanthoxyli). Some of these bacteria produce organic acids, potentially degrading stone. Actinobacteria, mainly streptomycetes, were present in most samples; algae and fungi were rare. A dark-pigmented filamentous fungus was detected in internal and external Preah Khan samples, while the alga Trentepohlia was found only in samples taken from external, pink-stained stone at Preah Khan. Results show that these microbial biofilms are mature communities whose major constituents are resistant to dehydration and high levels of irradiation and can be involved in deterioration of sandstone. Such analyses are important prerequisites to the application of control strategies.     

Characterization of biofilm growth and biocide susceptibility testing of Mycobacterium phlei using the MBEC assay system

The importance of non-tuberculosis mycobacterial biofilm species in medicine, industry and the environment has recently gained attention. Our objectives were to characterize biofilm growth of Mycobacterium phlei M4, as a model of rapidly growing mycobacteria using the minimal biofilm eradication concentration (MBEC) and to compare biocide susceptibility of planktonic and biofilm organisms. Scanning electron microscopy was also carried out to observe biofilm morphology. With the exception of Sporicidin and Virkon the minimum bactericidal concentration values for all biocides tested were lower than the MBEC values. The MBEC assay system was seen to produce multiple and reproducible biofilms of M. phlei and to be a useful tool for susceptibility studies.     

A simple in vitro model for growth control of bacterial biofilms

The perfused biofilm fermenter was found to be unsuitable for the long-term culture and growth rate control of Staphylococcus aureus and Pseudomonas aeruginosa biofilms. In a simplified approach, biofilms of these organisms were grown within Sorbarod filter plugs which were perfused with culture medium. Pseudo-steady states were established which were stable over several days at which the growth rate of the biofilm was reproducible, measurable and significantly slower than in broth culture. Environmental scanning electron microscopy of dissected Sorbarods demonstrated an association of cells with the surfaces of individual cellulose fibres, and growth characteristic of biofilms. Relatively high cell numbers recovered from the Sorbarod model facilitated biochemical investigations of biofilm populations and cells released spontaneously from them. SDS-PAGE demonstrated significant differences between the protein profiles of biofilm and eluted populations, which include, in Staph. aureus, the repression of a 48 kDa protein and increased expression of a 21 kDa protein relative to planktonic controls cultured at equivalent growth rates. The paper demonstrates the suitability of the approach for the culture of biofilm samples which are suitable for biochemical analysis.     

A simple in vitro model for growth control of bacterial biofilms

The perfused biofilm fermenter was found to be unsuitable for the long-term culture and growth rate control of Staphylococcus aureus and Pseudomonas aeruginosab biofilms. In a simplified approach, biofilms of these organisms were grown within Sorbarod filter plugs which were perfused with culture medium. Pseudo-steady states were established which were stable over several days at which the growth rate of the biofilm was reproducible, measurable and significantly slower than in broth culture. Environmental scanning electron microscopy of dissected Sorbarods demonstrated an association of cells with the surfaces of individual cellulose fibres, and growth characteristic of biofilms. Relatively high cell numbers recovered from the Sorbarod model facilitated biochemical investigations of biofilm populations and cells released spontaneously from them.
SDS-PAGE demonstrated significant differences between the protein profiles of biofilm and eluted populations, which include, in Staph. aureus , the repression of a 48 kDa protein and increased expression of a 21 kDa protein relative to planktonic controls cultured at equivalent growth rates. The paper demonstrates the suitability of the approach for the culture of biofilm samples which are suitable for biochemical analysis.     

Distribution of virus-like Particles in an Oligotrophic Marine Environment (Alboran Sea,Western Mediterranean)

Viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by one order of magnitude. In the present study, the spatial distribution of viruses in offshore waters of the Alboran Sea (Western Mediterranean) have been studied to determine the relationships between viruses and host communities in this oligotrophic marine environment. Viral abundance was determined using two methods: (i) epifluorescence light microscopy using the dsDNA binding fluorochrome DAPI, and (ii) direct counts by transmission electron microscopy (TEM). The results obtained were significantly different; the highest viral counts were obtained by mean of TEM analyses. In all the samples tested the number of viruses was exceeded by the bacterial concentrations, with a ratio between viral and bacterial titers varying between 1.4 and 20. VLP (virus-like particle) counts were not significantly correlated (p > 0.001) with chlorophyll a concentration or the abundance of cyanobacteria. However, there was a positive and significant correlation with bacterial abundance (p < 0.001). The analysis of size and morphology of viral particles by TEM and the correlation obtained between the numbers of VLP and bacteria suggest that the majority of the viral particles in the Alboran Sea are bacteriophages. None of the indirect evidence suggested that eukaryotic algae or cyanobacteria were important host organisms in these waters.     

The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk

Toxic cyanobacteria pose a significant hazard to human health and the environment. The recent characterisation of cyanotoxin synthetase gene clusters has resulted in an explosion of molecular detection methods for these organisms and their toxins. Conventional polymerase chain reaction (PCR) tests targeting cyanotoxin biosynthesis genes provide a rapid and sensitive means for detecting potentially toxic populations of cyanobacteria in water supplies. The adaptation of these simple PCR tests into quantitative methods has additionally enabled the monitoring of dynamic bloom populations and the identification of particularly problematic species. More recently, DNA microarray technology has been applied to cyanobacterial diagnostics offering a high-throughput option for detecting and differentiating toxic genotypes in complex samples. Together, these molecular methods are proving increasingly important for monitoring water quality.     

Photosynthetic pigment localization and thylakoid membrane morphology are altered in Synechocystis 6803 phycobilisome mutants

Cyanobacteria are oxygenic photosynthetic prokaryotes that are the progenitors of the chloroplasts of algae and plants. These organisms harvest light using large membrane-extrinsic phycobilisome antenna in addition to membrane-bound chlorophyll-containing proteins. Similar to eukaryotic photosynthetic organisms, cyanobacteria possess thylakoid membranes that house photosystem (PS) I and PSII, which drive the oxidation of water and the reduction of NADP+, respectively. While thylakoid morphology has been studied in some strains of cyanobacteria, the global distribution of PSI and PSII within the thylakoid membrane and the corresponding location of the light-harvesting phycobilisomes are not known in detail, and such information is required to understand the functioning of cyanobacterial photosynthesis on a larger scale. Here, we have addressed this question using a combination of electron microscopy and hyperspectral confocal fluorescence microscopy in wild-type Synechocystis species PCC 6803 and a series of mutants in which phycobilisomes are progressively truncated. We show that as the phycobilisome antenna is diminished, large-scale changes in thylakoid morphology are observed, accompanied by increased physical segregation of the two photosystems. Finally, we quantified the emission intensities originating from the two photosystems in vivo on a per cell basis to show that the PSI:PSII ratio is progressively decreased in the mutants. This results from both an increase in the amount of photosystem II and a decrease in the photosystem I concentration. We propose that these changes are an adaptive strategy that allows cells to balance the light absorption capabilities of photosystems I and II under light-limiting conditions.     

Observations on cyanobacterial population collapse in eutrophic lake water

In two laboratory-scale enclosures of water from the shallow, eutrophic Lake Loosdrecht (the Netherlands), the predominating filamentous cyanobacteria grew vigorously for 2 weeks, but then their populations simultaneously collapsed, whereas coccoid cyanobacteria and algae persisted . The collapse coincided with a short peak in the counts of virus-like particles. Transmission electron microscopy showed the morphotype Myoviridae phages, with isometric heads of about 90 nm outer diameter and >100-nm long tails, that occurred free, attached to and emerging from cyanobacterial cells. Also observed were other virus-like particles of various morphology. Similar mass mortality of the filamentous cyanobacteria occurred in later experiments, but not in Lake Loosdrecht. As applies to lakes in general, this lake exhibits high abundance of virus-like particles. The share and dynamics of infectious cyanophages remain to be established, and it is as yet unknown which factors primarily stabilize the host–cyanophage relationship. Observations on shallow, eutrophic lakes elsewhere indicate that the cyanophage control may also fail in natural water bodies exhibiting predominance of filamentous cyanobacteria. Rapid supply of nutrients appeared to be a common history of mass mortality of cyanobacteria and algae in laboratory and outdoor enclosures as well as in highly eutrophic lakes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cyanobacterial macrophytes in an Early Silurian (Llandovery) continental biota: Passage Creek, lower Massanutten Sandstone, Virginia, USA

A compression macrofossil with structure consisting of mineral-replaced filaments embedded in an amorphous carbonaceous matrix is described as a macrophytic cyanobacterial colony from continental assemblages of the Early Silurian (Llandovery) Passage Creek biota, in the lower Massanutten Sandstone (Virginia, USA). Filaments are predominantly multiseriate and consist of spheroidal crystalline aggregates representing early pyrite (subsequently replaced by iron hydroxides) precipitated preferentially inside cells. Interpretation of the fossils as cyanobacteria is based on close similarities to modern organisms in terms of overall morphology and production of copious extracellular investments, filament and cell sizes, and continental epigeal (freshwater or terrestrial) habitat. This interpretation incorporates data on cyanobacterial taphonomy and mechanisms of diagenetic mineral precipitation. These fossils are part of the oldest macrofossil assemblages documenting well-developed and diverse communities on continents. They provide the earliest direct evidence for cyanobacteria in strictly continental habitats, corroborating the commonly held but poorly documented view that cyanobacteria were among the initial colonizers of continents.     

Biological filtration limits carbon availability and affects downstream biofilm formation and community structure

Carbon removal strategies have gained popularity in the mitigation of biofouling in water reuse processes, but current biofilm-monitoring practices based on organic-carbon concentrations may not provide an accurate representation of the in situ biofilm problem. This study evaluated a submerged microtiter plate assay for direct and rapid monitoring of biofilm formation by subjecting the plates to a continuous flow of either secondary effluent (SE) or biofilter-treated secondary effluent (BF). This method was very robust, based on a high correlation (R(2) = 0.92) between the biomass (given by the A(600) in the microtiter plate assay) and the biovolume (determined from independent biofilms developed on glass slides under identical conditions) measurements, and revealed that the biomasses in BF biofilms were consistently lower than those in SE biofilms. The influence of the organic-carbon content on the biofilm community composition and succession was further evaluated using molecular tools. Terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed a group of pioneer colonizers, possibly represented by Sphingomonadaceae and Caulobacter organisms, to be common in both SE and BF biofilms. However, differences in organic-carbon availabilities in the two water samples eventually led to the selection of distinct biofilm communities. Alphaproteobacterial populations were confirmed by fluorescence in situ hybridization to be enriched in SE biofilms, while Betaproteobacteria were dominant in BF biofilms. Cloning analyses further demonstrated that microorganisms adapted for survival under low-substrate conditions (e.g., Aquabacterium, Caulobacter, and Legionella) were preferentially selected in the BF biofilm, suggesting that carbon limitation strategies may not achieve adequate biofouling control in the long run.     

Three-Dimensional Morphology, Ultrastructure, and Replication of Mycoplasma felis

The morphology and replication of Mycoplasma felis in relation to growth phase in culture were studied by electron microscopy. The organisms showed 1.0 to 1.45-hr doubling times with typical bacterial-type growth curves when grown in dialysate broth supplemented with horse serum. Organisms were fixed for electron microscopy by using Veronal acetate-buffered 0.8% OsO(4) (pH 6.1) in 20% sucrose. The morphology of exponential-phase organisms differed markedly from that of stationary or death-phase organisms, which were essentially large round forms with either dispersed or abnormally aggregated cytoplasm. Plasticine models prepared from serial sections of organisms in exponential phase showed the organisms to be either disc-shaped, triangular, horseshoe-shaped, or multilobular. A central "hole" was frequently present in these structures and could be visualized in the lobular forms as an interconnecting circular membrane. The inner surface of this membrane often showed contact with a small membranous body about 0.12 mum in diameter. The significance of this body is unknown. The morphology of the various shapes was confirmed by using the phosphotungstic acid and critical point methods. When the ratios of the various forms in exponential-phase cultures were determined, it was found that a replication sequence could be proposed which accounted for not only the volume increase required to accommodate deoxyribonucleic acid (DNA) replication but also the distribution of that DNA. Although it is likely that DNA replication in M. felis is a binary process, it appears that the mechanism for production of new cells need not be a binary process.     

CHROOCOCCOID CYANOBACTERIA IN LAKE ONTARIO: VERTICAL AND SEASONAL DISTRIBUTIONS DURING 19821

Chroococcoid cyanobacteria (0.7–1.3 μm in diameter) were discovered to be a significant component of the Lake Ontario plankton. Using epifluorescence microscopy, the densities of these microorganisms were found to vary by four orders of magnitude with a single large peak in abundance (6.5 × 10⁵ cells mL^?1) corresponding to the time of maximum water temperature. The morphology and abundance of these cyanobacteria were similar to those previously found in oceanic systems. They constituted 10% of the bacterial numbers in the epilimnion during this period, approximately 40% of the biomass of prokaryotes less than 2.0 μm, and 30% of the biomass of all microorganisms less than 20 μm in size. Size fractionation studies indicated that they were responsible for approximately 38% of the total primary production during times of peak abundance, and were important in phosphorus uptake. Cyanobacteria observed in the food vacuoles of heterotrophic microflagellates and in the guts of rotifers suggest that the latter organisms may be important consumers of this prokaryote population.     

Phototrophic biofilms and their potential applications

Phototrophic biofilms occur on surfaces exposed to light in a range of terrestrial and aquatic environments. Oxygenic phototrophs like diatoms, green algae, and cyanobacteria are the major primary producers that generate energy and reduce carbon dioxide, providing the system with organic substrates and oxygen. Photosynthesis fuels processes and conversions in the total biofilm community, including the metabolism of heterotrophic organisms. A matrix of polymeric substances secreted by phototrophs and heterotrophs enhances the attachment of the biofilm community. This review discusses the actual and potential applications of phototrophic biofilms in wastewater treatment, bioremediation, fish-feed production, biohydrogen production, and soil improvement.     

Mineralisation of filamentous cyanobacteria in Lake Thetis stromatolites,Western Australia

Stromatolites are cited as some of the earliest evidence for life on Earth, but problems remain in reconciling the paucity of microfossils in ancient carbonate examples with the abundance of microbes that help construct modern analogues. Here, we trace the mineralisation pathway of filamentous cyanobacteria within stromatolites from Lake Thetis, Western Australia, providing new insights into microfossil preservation in carbonate stromatolites. Lake Thetis cyanobacteria exhibit a spectrum of mineralisation processes that include early precipitation of Mg‐silicates, largely controlled by the morphochemical features of the cyanobacteria, followed by aragonite formation that is inferred to be driven by heterotrophic activity. Fossilised cyanobacteria with high‐quality morphological preservation are characterised by a significant volume of authigenic Mg‐silicates, which have preferentially nucleated in/on extracellular organic material and on cell walls, and now replicate the region once occupied by the cyanobacterial sheath. In such specimens, aragonite is restricted to the outer sheath margin and parts of the cell interior. Cyanobacteria that display more significant degradation appear to possess a higher ratio of aragonite to Mg‐silicate. In these specimens, aragonite forms micronodules in the sheath zone and is spatially associated with the inferred remains of heterotrophic bacteria. Aragonite also occurs as an advancing front from the outer margin of the sheath where it is commonly intergrown with Mg‐silicates. Where there is no evidence of Mg‐silicates within filaments, the fidelity of microfossil preservation is poor. In these cases, individual filaments may no longer be visible under light microscopy, and little organic material remains, but filament traces remain detectable using electron microscopy due to variations in aragonite texture. These data provide further evidence that authigenic silicate minerals play a crucial role in the fossilisation of micro‐organisms; in their absence, carbonate crystal growth potentially mediated by heterotrophic microbial decay may largely obliterate morphological evidence for life within stromatolites, although mineralogical traces may still be detectable using electron microscopy.