Cyanobacterial Biofilms Research Articles

Novel members of the family Flavobacteriaceae from Antarctic maritime habitats including Subsaximicrobium wynnwilliamsii gen. nov., sp. nov., Subsaximicrobium saxinquilinus sp. nov., Subsaxibacter broadyi gen. nov., sp. nov., Lacinutrix copepodicola gen. nov., sp. nov., and novel species of the genera Bizionia, Gelidibacter and Gillisia

Several orange- and yellow-pigmented, halophilic, strictly aerobic, chemoheterotrophic, Gram-negative strains were isolated during investigations of maritime Antarctic habitats, including coastal fast sea-ice brine and algae, crustaceans and quartz stone sublithic cyanobacterial biofilms. Isolates investigated in this study belonged to the marine clade of the family Flavobacteriaceae and represented lineages that were either distinct from species with validly published names or appeared to be distinct species within existing genera. A polyphasic taxonomic analysis demonstrated the novelty of these strains, and several new taxa are proposed. Strains from quartz stone sublithic communities were grouped into two new genera designated Subsaximicrobium gen. nov. and Subsaxibacter gen. nov. The genus Subsaximicrobium included the species Subsaximicrobium wynnwilliamsii sp. nov. (type species; type strain G#7(T)=ACAM 1070(T)=CIP 108525(T)) and Subsaximicrobium saxinquilinus sp. nov. (type strain Y4-5(T)=ACAM 1063(T)=CIP 108526(T)). The genus Subsaxibacter contained a single species designated Subsaxibacter broadyi sp. nov. (type strain P7(T)=ACAM 1064(T)=CIP 108527(T)). A novel bacterial strain isolated from the lake-dwelling, calanoid copepod Paralabidocera antarctica was given the name Lacinutrix copepodicola gen. nov., sp. nov. (type strain DJ3(T)=ACAM 1055(T)=CIP 108538(T)). Four novel species of the genus Bizionia were discovered, Bizionia algoritergicola sp. nov. (type strain APA-1(T)=ACAM 1056(T)=CIP 108533(T)) and Bizionia myxarmorum sp. nov. (type strain ADA-4(T)=ACAM 1058(T)=CIP 108535(T)), which were isolated from the carapace surfaces of sea-ice algae-feeding amphipods, and Bizionia gelidisalsuginis sp. nov. (type strain IC164(T)=ACAM 1057(T)=CIP 108536(T)) and Bizionia saleffrena sp. nov. (type strain HFD(T)=ACAM 1059(T)=CIP 108534(T)), which were isolated from sea-ice brines. Several other novel species were also isolated from sea-ice samples, including two novel species of the genus Gelidibacter, Gelidibacter gilvus sp. nov. (type strain IC158(T)=ACAM 1054(T)=CIP 108531(T)) and Gelidibacter salicanalis sp. nov. (type strain IC162(T)=ACAM 1053(T)=CIP 108532(T)), as well as three novel species of the genus Gillisia, Gillisia illustrilutea sp. nov. (type strain IC157(T)=ACAM 1062(T)=CIP 108530(T)), Gillisia sandarakina sp. nov. (type strain IC148(T)=ACAM 1060(T)=CIP 108529(T)) and Gillisia hiemivivida sp. nov. (type strain IC154(T)=ACAM 1061(T)=CIP 108528(T)).

Insecticidal compounds of the biofilm‐forming cyanobacterium Fischerella sp. (ATCC 43239)

Cyanobacterial biofilms are grazed by many different benthic invertebrates. In particular, larvae of insects are often present in great numbers and exert strong grazing pressure on cyanobacteria. Along with morphological adaptations, allelochemicals may have been developed as defense mechanisms against insect larvae. To investigate the chemical defense of biofilm-forming cyanobacteria, larvae of Chironomus sp., a widely distributed genus in this habitat, were used. Ten artificial biofilms of axenic and nonaxenic cyanobacteria were screened for insecticidal activity against Chironomus sp. Fischerella 43239 was the cyanobacterium that exhibited the highest acute toxicity. A bioassay-guided isolation procedure was used to study the compounds responsible for toxicity in more detail. A toxic fraction was obtained when the 60% methanolic extract of Fischerella 43239 was separated on a C18 HPLC column. Electrospray mass spectrometry indicated the presence of several compounds in this fraction. The successful separation into individual compounds was achieved by HPLC on a cyanopropyl column. The heavily clustered quasimolecular ions observed on an electrospray mass spectrometer and the absorption spectra of the separated compounds were indicative of indole derivatives. The existence in the benthic cyanobacterium Fischerella 43239 of strong insecticidal metabolites that serve as chemical protection agents against insect larvae is supported by the data.

Flow analysis techniques for phosphorus: an overview

A bibliographical review on the implementation and the results obtained in the use of different flow analytical techniques for the determination of phosphorus is carried out. The sources, occurrence and importance of phosphorus together with several aspects regarding the analysis and terminology used in the determination of this element are briefly described. A classification as well as a brief description of the basis, advantages and disadvantages of the different existing flow techniques, namely; segmented flow analysis (SFA), flow injection analysis (FIA), sequential injection analysis (SIA), all injection analysis (AIA), batch injection analysis (BIA), multicommutated FIA (MCFIA), multisyringe FIA (MSFIA) and multipumped FIA (MPFIA) is also carried out. The most relevant manuscripts regarding the analysis of phosphorus by means of flow techniques are herein classified according to the detection instrumental technique used with the aim to facilitate their study and obtain an overall scope. Finally, the analytical characteristics of numerous flow-methods reported in the literature are provided in the form of a table and their applicability to samples with different matrixes, namely water samples (marine, river, estuarine, waste, industrial, drinking, etc.), soils leachates, plant leaves, toothpaste, detergents, foodstuffs (wine, orange juice, milk), biological samples, sugars, fertilizer, hydroponic solutions, soils extracts and cyanobacterial biofilms are tabulated.

Odor compounds from cyanobacterial biofilms acting as attractants and repellents for free‐living nematodes

Nematodes can both taste and smell an array of compounds, but whether and how these senses effect their capacity to locate microhabitats in aquatic environments is not known. Cyanobacterial biofilms may offer structure, shelter, and food for nematodes and are known to produce a variety of odor compounds. We studied the chemotaxis response of the freshwater nematode Bursilla monhystera (Rhabditidae) and the terrestrial model organism Caenorhabditis elegans (Rhabditidae) to odors of cyanobacterial biofilms. We used gas chromatography‐mass spectometry ultra‐trace analysis to identify odor compounds produced by two epilithic cyanobacterial biofilms of Lake Zurich. We also studied artificial, axenic biofilms of Plectonema sp., Calothrix sp., and Calothrix parietina, to assign these compounds to the metabolism of the cyanobacteria. The axenic cyanobacteria and epilithic biofilms had many odor compounds in common. B. monhystera was significantly attracted to Plectonema sp. and C. parietina but not to Calothrix sp. C. elegans, in contrast, was not attracted to any of these cyanobacterial biofilms. Furthermore, we applied a multicomponent mixture of odor compounds and found significant attraction for both nematodes. Although C. elegans was also attracted by a variety of single odor compounds, B. monhystera was not attracted to any of the volatiles tested. β‐ionone even repelled this species. Our experiments demonstrate that aquatic nematodes are attracted to cyanobacterial biofilms using odor compounds as chemical cues. In contrast to the model organism, C. elegans, the chemotaxis of the aquatic nematode is elicited by a multicomponent odor rather than by single compounds.

Investigation of amperometric detection of phosphate: Application in seawater and cyanobacterial biofilm samples

Detection of phosphate using amperometry was investigated. The phosphomolybdate complex, formed by addition of nitric acid, ammonium molybdate and phosphate, was reduced at a carbon paste electrode polarised at +0.3V (versus Ag/AgCl). The major characteristics observed were simplicity of the equipment, a limited consumption of reagents and a low detection limit (0.3μmoll−1), with a linear range between 1 and 20μmoll−1. The interference of silicate was completely eliminated using an appropriate concentration of nitric acid and ammonium molybdate.The amperometric detection of orthophosphate in seawater using the batch injection analysis (BIA) technique was reported.Moreover, a carbon paste microelectrode was constructed. Its use allows the analysis of small volume of samples with little dilution in supporting electrolyte. This method was applied to the determination of orthophosphate in cyanobacterial biofilms collected from Roman catacombs. There was a good statistical correlation between results obtained with the proposed method and the standard spectrophotometric method.

Algal and cyanobacterial biofilms on calcareous historic buildings.

Major microorganisms in biofilms on external surfaces of historic buildings are algae, cyanobacteria, bacteria, and fungi. Their growth causes discoloration and degradation. We compared the phototrophs on cement-based renderings and limestone substrates at 14 historic locations (47 sites sampled) in Europe and Latin America. Most biofilms contained both cyanobacteria and algae. Single-celled and colonial cyanobacteria frequently constituted the major phototroph biomass on limestone monuments (32 sites sampled). Greater numbers of phototrophs, and especially of algae and of filamentous morphotypes, were found on cement-based renderings (15 sites), probably owing to the porosity and small pore size of the latter substrates, allowing greater entry and retention of water. All phototrophic groups were more frequent on Latin American than on European buildings (20 and 27 sites, respectively), with cyanobacteria and filamentous phototrophs showing the greatest differences. The results confirm the influence of both climate and substrate on phototroph colonization of historic buildings.

ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF EXTRACELLULAR POLYMERIC SECRETIONS (EPS) FROM MODERN SOFT MARINE STROMATOLITES (BAHAMAS) AND ITS INHIBITORY EFFECT ON CACO3 PRECIPITATION

ABSTRACT Bahamian soft marine stromatolites consist of cyanobacterial biofilms and carbonate sand grains (ooids) embedded in their extracellular polymeric secretions (EPS). EPS were isolated from natural marine stromatolites and the laboratory cultured stromatolite forming cyanobacterium isolateSchizothix sp. Laboratory investigations were conducted to examine biochemical characteristics and the role of EPS in the inhibition of CaCO3 precipitation. EPS consisted of acid polysaccharides and proteins. SDS-PAGE and amino acid analysis suggested that EPS from both soft marine stromatolite and Schizothrix sp. mat contained small proteins (38 kD and 45 kD) enriched in aspartic acid and glutamic acid. Also, immuno blotting suggested that natural EPS contain high molecular weight acid polysaccharide (500 k) which may represent cross-linked products of laboratory cultured Schizothrix sp. acid polysaccharide (300 k). EPS from both soft marine stromatolite and laboratory cultured Schizothrix sp. inhibited CaCO3 precipitation in vitro, as determined using pH drift assays examining pH decrease which occur in response to CaCO3 precipitation. PH drift assays of enzymatically and chemically modified EPS isolated from soft marine stromatolite and laboratory cultured Schizothrix sp. indicated that both uronic acids and protein fractions may be involved in the inhibition of CaCO3 precipitation.

Photosynthesis-induced biofilm calcification and calcium concentrations in Phanerozoic oceans.

Photosynthetic carbon assimilation is commonly invoked as the cause of calcium carbonate precipitation in cyanobacterial biofilms that results in the formation of calcareous stromatolites. However, biofilm calcification patterns in recent lakes and simulation of photosynthetically induced rise in calcium carbonate supersaturation demonstrate that this mechanism applies only in settings low in dissolved inorganic carbon and high in calcium. Taking into account paleo-partial pressure curves for carbon dioxide, we show that Phanerozoic oceans sustaining calcified cyanobacteria must have had considerably higher calcium concentrations than oceans of today. In turn, the enigmatic lack of calcified cyanobacteria in stromatolite-bearing Precambrian sequences can now be explained as a result of high dissolved inorganic carbon concentrations.

Evidence of allelochemical activity in subtropical cyanobacterial biofilms of Taiwan

Cyanobacterial biofilms were collected in the subtropical part of Taiwan from wet and irrigated rocks, stone and concrete walls, and drains. The biofilms developed in unpolluted freshwater and consisted of various genera, including Phormidium, Lyngbya, Oscillatoria, Microcoleus, Aphanothece and Scytonema. Ethanolic extracts of these biofilms were used to test for growth inhibition activity against cyanobacteria (Anabaena 7120 and Anabaena P-9), grazer toxicity (acute toxicity of Thamnocephalus platyurus) and inhibition activity of extracellular and ectoenzymatic polysaccharide hydrolysis (a-glucosidase inhibition). More than 20 % of the extracts were active in the different bioassays. Most Phormidium biofilms showed growth inhibition activity; the strongest grazer toxicity was found in a Nostoc biofilm, while a Scytonema biofilm exhibited the strongest inhibitor activity against α-glucosidase. The activities shown in the different bioassays were not accumulated in a particular biofilm but were distributed over different collections and demonstrate the wide occurrence and importance of allelochemistry in cyanobacterial biofilms. The growth inhibition test for cyanobacteria was miniaturized to allow the assay of very small amounts of growth inhibitory extracts.

Separation and preconcentration of copper and cadmium ions from multielemental solutions using Nostoc muscorum-based biosorbents

Nostoc-based biosorbents (AlgaSORBs) useful as chromatographic column-packing materials were prepared by immobilizing cyanobacteria onto solid support in three different fashions: (i) cyanobacterial biofilm (Nostoc-dimethylformamide slurry) over polymer-modified silica gel, (ii) cyanobacterial biofilm over bare silica gel, and (iii) cyanobacteria as such onto polymer-modified silica gel. The materials were characterized for their stabilities and metal sorption/elution conditions under static and dynamic equilibrations. Preconcentrated metals from a test sample were detected following `standard addition' method using a differential pulse anodic stripping voltammetric technique. All sorbents showed 100% affinity for Cd2+ ion in a multielemental sample at pH 6.9 and a flow rate of 0.5 ml/min with a preconcentration factor varying between 28- and 75-fold. The first type of AlgaSORB was also found to be selective for Cu2+ ion in multielemental analysis at pH 5.2 and a flow rate of 1.0 ml/min with a preconcentration factor of 75. The low capacity and favourable kinetics of these sorbents for Cu2+, Cd2+, Zn2+ and Pb2+ ions reflect the suitability of AlgaSORB columns for satisfactory performance in single column ion-chromatography. The polymer spacer between cyanobacterial biofilm and silica gel plays a vital role in holding the immobilized biofilm resulting in better endurance and recyclability for the first type of biosorbent.

Effect of photosynthesis on pH variation in cyanobacterial biofilms from Roman catacombs

Cyanobacterial biofilms present on stone surfaces inRoman hypogea were studied with the aim of assessingtheir deteriogenic activity on the colonisedsubstrata. In order to achieve this, non-destructivemethods were developed and applied to measure pHvariation induced via photosynthesis and respirationin representative cyanobacteria from Roman catacombs.Amperometric and potentiometric microsensors were alsoused on Scytonema biofilms in culture in orderto measure photosynthesis and assess pH decreases andincreases during dark–light periods. Measurementsof pH showed that, starting with values slightly belowneutral, the pH in Scytonema biofilms increasedby 0.24–0.77 units in the transition from dark to1000 μmol photon m-2 s-1 irradiance.Comparison of photosynthesis and pH curves recordedsimultaneously on the same artificial biofilm showeda maximum increase in pH value at irradiances higherthan those saturating photosynthesis. Alkalinisationof the substrate during illumination occurred to asufficient extent to induce precipitation of mineralcompounds, especially on calcareous substrates such asthose present in Roman hypogea.

Calcification in cyanobacterial biofilms of alkaline salt lakes

Geomicrobiological analysis of calcifying biofilms of three alkaline salt lakes characterized by moderate to high carbonate alkalinity indicates that microbial carbonate rock formation is not directly linked to cyanobacterial carbon fixation. The present review summarizes results from two published case studies that have been carried out at Pyramid Lake, USA, and Lake Nuoertu, PR China. New observations and data are presented for a current project on Satonda Crater Lake, Indonesia, that revise previous conclusions concerning the relationship between cyanobacteria and biofilm calcification. Extracellular polymeric substances (EPS) in the investigated lakes are mostly produced by cyanobacteria; their properties are discussed as key factors in biofilm calcification. In particular, EPS are capable of binding divalent cations (e.g. Ca2+) from the liquid phase by their carboxylate and sulphate groups. Therefore, despite a high supersaturation of the lake water with respect to calcium carbonate minerals, precipitation does not take place immediately. A delayed onset of precipitation can be achieved by a continuous Ca2+ supply that exceeds the Ca2+-binding capacity of the EPS, and/or an exoenzymatic degradation (decarboxylation, cleavage) of mucous substances that reduces the binding capacity and causes secondary Ca2+ release. The resulting microcrystalline precipitates are randomly distributed within the EPS, usually away from any of the living cyanobacteria. This suggests that the effect of photosynthetic CO2 fixation in increasing supersaturation is of secondary importance at high alkalinities. In contrast to biofilm-covered surfaces, calcium carbonate minerals nucleate and grow rapidly at surfaces poor in EPS when the critical supersaturation level for non-enzymatically controlled carbonate precipitation is reached. Examples of such surfaces poor in EPS are dead, lysed green algal cells and thin, discontinuous biofilms in voids of microbial reef rocks. Calcium carbonate crystals directly linked to cyanobacterial cells or filaments have been observed only exceptionally, e.g. on Calothrix.

Calcification in cyanobacterial biofilms of alkaline salt lakes

Geomicrobiological analysis of calcifying biofilms of three alkaline salt lakes characterized by moderate to high carbonate alkalinity indicates that microbial carbonate rock formation is not directly linked to cyanobacterial carbon fixation. The present review summarizes results from two published case studies that have been carried out at Pyramid Lake, USA, and Lake Nuoertu, PR China. New observations and data are presented for a current project on Satonda Crater Lake, Indonesia, that revise previous conclusions concerning the relationship between cyanobacteria and biofilm calcification. Extracellular polymeric substances (EPS) in the investigated lakes are mostly produced by cyanobacteria; their properties are discussed as key factors in biofilm calcification. In particular, EPS are capable of binding divalent cations (e.g. Ca2+) from the liquid phase by their carboxylate and sulphate groups. Therefore, despite a high supersaturation of the lake water with respect to calcium carbonate minerals, precipitation does not take place immediately. A delayed onset of precipitation can be achieved by a continuous Ca2+ supply that exceeds the Ca2+-binding capacity of the EPS, and/or an exoenzymatic degradation (decarboxylation, cleavage) of mucous substances that reduces the binding capacity and causes secondary Ca2+ release. The resulting microcrystalline precipitates are randomly distributed within the EPS, usually away from any of the living cyanobacteria. This suggests that the effect of photosynthetic CO2 fixation in increasing supersaturation is of secondary importance at high alkalinities. In contrast to biofilm-covered surfaces, calcium carbonate minerals nucleate and grow rapidly at surfaces poor in EPS when the critical supersaturation level for non-enzymatically controlled carbonate precipitation is reached. Examples of such surfaces poor in EPS are dead, lysed green algal cells and thin, discontinuous biofilms in voids of microbial reef rocks. Calcium carbonate crystals directly linked to cyanobacterial cells or filaments have been observed only exceptionally, e.g. on Calothrix.

Cyanobacterial biofilms in sewage treatment plants along the Thyrrenian coast (Mediterranean Sea), Italy

Cyanobacterial biofilms in sewage treatment plants along the Thyrrenian coast (Mediterranean Sea), Italy

Biofilm exopolymers control microbialite formation at thermal springs discharging into the alkaline Pyramid Lake, Nevada, USA

Calcium carbonate precipitation and microbialite formation at highly supersaturated mixing zones of thermal spring waters and alkaline lake water have been investigated at Pyramid Lake, Nevada. Without precipitation, pure mixing should lead to a nearly 100-fold supersaturation at 40oC. Physicochemical precipitation is modified or even inhibited by the properties of biofilms, dependent on the extent of biofilm development and the current precipitation rate. Mucus substances (extracellular polymeric substances, EPS, e.g., of cyanobacteria) serve as effective Ca 2C -buffers, thus preventing seed crystal nucleation even in a highly supersaturated macroenvironment. Carbonate is then preferentially precipitated in mucus-free areas such as empty diatom tests or voids. After the buffer capacity of the EPS is surpassed, precipitation is observed at the margins of mucus areas. Hydrocarbon biomarkers extracted from (1) a calcifying Phormidium-biofilm, (2) the stromatolitic carbonate below, and (3) a fossil ‘tufa’ of the Pleistocene pinnacles, indicate that the cyanobacterial primary producers have been subject to significant temporal changes in their species distribution. Accordingly, the species composition of cyanobacterial biofilms does not appear to be relevant for the formation of microbial carbonates in Pyramid Lake. The results demonstrate the crucial influence of mucus substances on carbonate precipitation in highly supersaturated natural environments. © 1999 Elsevier Science B.V. All rights reserved.

Long-term effects of UV and visible irradiance on natural populations of a scytonemin-containing cyanobacterium ( Calothrix sp.)

A natural warm spring stream with a cyanobacterial biofilm community composed almost entirely of Calothrix sp., a species rich in the UV-absorbing sheath pigment scytonemin, was used for 3 month summer experiments to test the following hypotheses: (1) UV radiation is necessary for the synthesis of high scytonemin content in sheaths. (2) High scytonemin content is required for uninhibited photosynthesis under high UV flux. Both of these hypotheses were answered affirmatively in earlier experiments with laboratory cultures of other cyanobacterial species. However, the tests of these hypotheses under natural conditions were necessary to confirm these conclusions, mainly because fluxes of UV and visible radiation and their ratios are very different under field conditions. Intact mats of Calothrix, siliceous substrate cleared of Calothrix, and artificial foam were treated for 3 months under filters transparent to UV radiation and visible light, and under filters that excluded only UV radiation. Neutral density screens were used in combination with filters to reduce the total irradiance over some sections of the stream. After 3 months, under all treatments, the predominant organism was still the same morphotype of Calothrix. Intact Calothrix mats produced high levels of scytonemin over the summer, except under low UV and low visible irradiance, while chlorophyll-a values per unit area remained relatively low and showed little change under all treatments. After 3 months, intact Calothrix mat and colonized mat with substantial scytonemin were not inhibited photosynthetically by UV irradiance. Only colonized populations exposed to ∼12% UV and ∼12% visible irradiance, and with very low scytonemin content, showed significant UV-inhibition. Thus, the correlation between UV protection and scytonemin presence was established experimentally for the first time under solar irradiance in a natural, essentially monospecific population of a cyanobacterium.

On the method of integration of tidal equations suggested by Fairbrain. : Stretenski, L.N. 1962 Izv. Akad. Nauk, USSR, Geofiz. S. (7): 947–954. 7: 604–609.

Many archaeologically important stone temples, caves, mortar monuments with artistic expression as well as building facades of India are now disfigured due to colonization of cyanobacterial biofilms leading to weathering of the substratum. They are composed of species principally belonging to the genera Hassallia, Tolypothrix, Scytonema, Lyngbya and Calothrix, which appeared soon after wetting of the biofilms. Several other species of genera Aulosira, Nostoc, Camptylonema, Dichothrix, Chlorogloeopsis and Westiellopsis occurred as associated organisms as they appeared upon prolonged culture of the biofilms. Molecular phylogenetic analysis based on 16S rRNA partial gene sequencing of all these 24 cyanobacteria species under 11 genera isolated from the surfaces of monuments and building facades of India along with those of other species isolated from stone surfaces in subaerial habitats and hypogeal environments in different regions of the globe showed that those species from tropical regions were clustered together, quite different from cyanobacteria of the genera Chroococcidiopsis, Leptolyngbya, Phormidium, Nostoc, Rexia, Symphyonemopsis, Scytonema, Tolypothrix and Calothrix occurring in the temperate regions.