Purification of biosilica from living diatoms by a two-step acid cleaning and baking method

Due to their sustainability, intact cell walls, availability of pure cultures, and others, living diatoms show a lot of promise for the application in various fields in particular for micro/nano-devices. In order to purify the biosilica structures of diatoms called frustules, a two-step acid cleaning and baking method was employed. By this path, organic matter and inorganic impurities can be removed very effectively. In addition, the highest quality of frustules was achieved when the samples were cleaned in an excess of boiling 10~15 % HCl and subsequently heated to 600 °C at a heating rate of 3 °C min^?1 for 6 h. In our operation, the native frustule morphology was maintained completely, and dry frustules with more than 90 % SiO₂ in weight can be obtained, and furthermore, the surface area of them reached a good value of 48.47 m² g^?1.     

VALVE AND BAND MORPHOLOGY OF SOME FRESHWATER DIATOMS. IV. OUTER SURFACE MUCILAGE OF NAVICULA CONFERVACEA VAR. CONFERVACEA1

The development of the mucilage on the outer surface of Navicula confervacea (Kütz.) Grun., a raphed, filamentous diatom, was studied with scanning electron microscopy. This nonstructural cell wall material, present on the surface after critical-point drying and absent after acid cleaning, was of two types: strands and papillae. Strands were associated with the raphe system, areolae, elongated pores of the mantle, and all girdle sutures. Organic papillae were a common feature of valves, valvo-copulae and pleurae, but their origin and distribution could not be explained since they often occurred between the obvious openings in the frustule. Strands from the raphe and areolae may function in attaching terminal cells to a substrate and adjacent cells to each other. Other strands of the girdle arise from sutures during cell enlargement and continue to lengthen and intertwine until the individual frustules within a filament are obscured. Strands from sutures might originate from the advalvar row of pores of the girdle bands since these pores lie along the suture, but direct observation of this was not made. Secretion between, the bands also cannot be ruled out. Although mucilaginous papillae may sometimes occur at random on the entire surface of frustules, there is also a distinct, narrow multiseriate row of them around the edge of valves without marginal spines.     

Processing samples of benthic marine diatoms from Mediterranean oligotrophic areas

The processing of benthic diatoms is tedious and involves several potentially damaging steps for cells. Although the preservation of siliceous frustules is of paramount importance in the implementation of biotic indices, only few studies quantified treatment-induced cell losses. We assumed that commonly used treatments may lead to mechanical (centrifugation, sedimentation, boiling, sonication and mounting in Naphrax) and chemical (cold H₂O₂ digestion) damages on diatoms. We analysed the potential adverse effects of these treatments and the cleaning efficiency of H₂O₂ and incineration in order to find out the most suitable technique to process lightly silicified Mediterranean populations. Results showed that successive resuspensions of material after each concentration treatment (sedimentation and centrifugation) and low speed centrifugation did not alter the physical integrity of frustules. In contrast, boiling and sonication exhibited adverse effects especially on the preservation of large frustules and Naphrax mounting proved to be the most damaging step whatever the size of diatoms. For cleaning treatments, incineration provided the most satisfactory results and acted on a non-selective way as opposed to hydrogen peroxide which led to either a large number of non-cleaned frustules or dissolved valves. Our recommendations for processing samples of lightly silicified Mediterranean benthic diatoms include the use of low-speed centrifugations, dehydration at room temperature, incineration and dry mounting.     

EVIDENCE OF AN ORGANIC MATRIX FROM DIATOM BIOSILICA1

Most biominerals appear to be composites of organic material and mineral. Whether biosilica is such a composite is unresolved because of a lack of evidence for such organic components. We present evidence that organic material exists within diatom biosilica and can be extracted using HF/NH₄F solutions from frustules isolated from Cyclotella meneghiniana Kütz and diatomaceous earth. To eliminate organic casing on the silicified frustules as a source of organic materials, the casing was removed by oxidation of frustules with NaOCl before extraction. The removal of the casing was confirmed in that oxidized frustules no longer displayed the ability to be stained with ruthenium red and fluorescamine. Frustules examined with EDXA showed an emission peak from sulfur before treatment but no peak following treatment, indicating that oxidation removed organic sulfur. The organic material obtained from extracts of fresh frustules contained both soluble and insoluble components. Only soluble material was evident in extracts from diatomaceous earth. The soluble material appears to contain glycoproteins with relatively high levels of serine and glycine. The soluble proteins from fresh frustules also appear to be phosphorylated. Indirect evidence is presented that suggests the soluble proteins may contain regions of primary structure enriched in anionic amino acids. The soluble extracts differ from general cell contents when the two fractions are compared, suggesting that frustules contain specialized organic material. The identification of silica-specific organic material suggests that mineralization in diatoms may be in part matrix-mediated.     

Preparation of biosilica structures from frustules of diatoms and their applications: current state and perspectives

Frustules, the silica shells of diatoms, have unique porous architectures with good mechanical strength. In recent years, biologists have learned more about the mechanism of biosilica shells formation; meanwhile, physicists have revealed their optical and microfluidic properties, and chemists have identified ways to modify them into various materials while maintaining their hierarchical structures. These efforts have provided more opportunities to use biosilica structures in microsystems and other commercial products. This review focuses on the preparation of biosilica structures and their applications, especially in the development of microdevices. We discuss existing methods of extracting biosilica from diatomite and diatoms, introduce methods of separating biosilica structures by shape and sizes, and summarize recent studies on diatom-based devices used for biosensing, drug delivery, and energy applications. In addition, we introduce some new findings on diatoms, such as the elastic deformable characteristics of biosilica structures, and offer perspectives on planting diatom biosilica in microsystems.     

Marine diatoms as optical biosensors

We have chemically modified the frustules of the marine diatom Coscinodiscus concinnus Wm. Smith to properly bind a highly selective bioprobe such as an antibody. By measuring the changes in the photoluminescence emission of diatoms frustules, we have monitored the molecular recognition event between the antibody and its ligand: the dissociation constant estimated is of the same order of that measured by standard Biacore. The nanostructured silica frustules, a low-cost and natural available material, have shown high sensitivity, equal to 1.2+/-0.2 nm microM(-1), and a detection limit of 100 nM, and thus are quite ideal candidates for lab-on-particle applications.     

Ultra‐sensitive immunoassay biosensors using hybrid plasmonic‐biosilica nanostructured materials

We experimentally demonstrate an ultra‐sensitive immunoassay biosensor using diatom biosilica with self‐assembled plasmonic nanoparticles. As the nature‐created photonic crystal structures, diatoms have been adopted to enhance surface plasmon resonances of metal nanoparticles on the surfaces of diatom frustules and to increase the sensitivity of surface‐enhanced Raman scattering (SERS). In this study, a sandwich SERS immunoassay is developed based on the hybrid plasmonic‐biosilica nanostructured materials that are functionalized with goat anti‐mouse IgG. Our experimental results show that diatom frustules improve the detection limit of mouse IgG to 10 pg/mL, which is ?100× better than conventional colloidal SERS sensors on flat glass.

Ultra‐sensitive immunoassay biosensor using diatom biosilica with self‐assembled plasmonic nanoparticles.     

Frustule morphogenesis of raphid pennate diatom <Emphasis Type="Italic">Encyonema ventricosum</Emphasis> (Agardh) Grunow

Diatoms stand out among other microalgae due to the high diversity of species-specific silica frustules whose components (valves and girdle bands) are formed within the cell in special organelles called silica deposition vesicles (SDVs). Research on cell structure and morphogenesis of frustule elements in diatoms of different taxonomic groups has been carried out since the 1950s but is still relevant today. Here, cytological features and valve morphogenesis in the freshwater raphid pennate diatom Encyonema ventricosum (Agardh) Grunow have been studied using light and transmission electron microscopy of cleaned frustules and ultrathin sections of cells, and scanning electron and atomic force microscopy of the frustule surface. Data have been obtained on chloroplast structure: the pyrenoid is spherical, penetrated by a lamella (a stack of two thylakoids); the girdle lamella consists of several short lamellae. The basic stages of frustule morphogenesis characteristic of raphid pennate diatoms have been traced, with the presence of cytoskeletal elements near SDVs being observed throughout this process. Degradation of the plasmalemma and silicalemma is shown to take place when the newly formed valve is released into the space between sister cells. The role of vesicular transport and exocytosis in the gliding of pennate diatoms is discussed.     

MORPHOLOGICAL VARIATION,SPECIES CONCEPTS,AND CLASSIFICATION OF AN UNUSUAL FOSSIL CENTRIC DIATOM (BACILLARIOPHYTA) FROM WESTERN NORTH AMERICA1

The classification system of diatoms (Bacillariophyta) is based almost entirely on the organization and structure of their silica cell walls. We have discovered Miocene freshwater fossils that contain valves of two different orders within the same organism. Within frustules of the genus Ellerbeckia Crawford, a genus of the order Melosirales, are contained frustules of the genus Actinocyclus Ehrenberg, a member of the Coscinodiscales. Based on the abundance of each morphotype, they appear to be able to reproduce themselves. The ability of a single diatom taxon to express this degree of morphological variability is unprecedented. These results suggest species concepts in diatoms may be in need of review and challenge the basis of the classification system for this widely distributed and ecologically important group of organisms.     

Chemical modification of Nitzschia panduriformis's frustules for protein and viral nanoparticle adsorption

The frustule of diatoms, through appropriate chemical modification, can be developed for a high adsorption level of recombinant proteins and viral nanoparticles. Field emission scanning electron microscopy (FE-SEM) analysis of clean frustules revealed a 3D loculate areolae structure (valvar phase porous pattern of the siliceous cell wall). Isocyanatopropyl triethoxysilane (IPS) and iminodiacetic acid (IDA) were used to immobilize Cu²⁺ ions (an average Cu²⁺ adsorption capacity about 190 μmol of Cu²⁺/ml of the Cu²⁺-coupled biosilica reached). FE-SEM, energy dispersion X-ray spectroscopy (EDS) and Fourier transform infrared (FT-IR) were used to confirm the chemical modification of the Cu²⁺-coupled biosilica. Protein adsorption was confirmed with the detection of a recombinant (His)₆-tagged green fluorescent protein binding using fluorescent microscopy. Infectious bursal disease virus VP2-441 subviral particles (SVPs) were found to bind to the Cu²⁺-coupled biosilica (approximately 3 × 10^?9 mol of VP2-441 SVPs/ml of modified frustules), a level higher than the previously obtained 9 × 10^?10 mol/ml for SVP binding using a commercial Ni–NTA resin. These give diatom frustules the potential to be developed into a material useful in viral nanoparticle purification systems or as a biosensor for the detection of viruses.     

Dissection of the frustules of the diatom Synedra acus under the action of picosecond impulses of submillimeter laser irradiation

Diatom algae realize highly intriguing processes of biosynthesis of siliceous structures in living cells under moderate conditions. Investigation of diatom physiology is complicated by frustule (siliceous exoskeleton). Frustules consist of valves and girdle bands which are adhered to each other by means of organic substances. Removal of the frustule from the lipid membrane of diatom cells would open new possibilities for study of silicon metabolism in diatoms. We found that submillimeter laser irradiation produced by a free-electron laser causes splitting of diatom frustules without destruction of cell content. This finding opens the way to direct study of diatom cell membrane and to isolation of cell organelles, including silica deposition vesicles. We suppose that the dissection action of the submillimeter irradiation results from unusual ultrasonic waves produced by the short (30–100 ps) but high-power (1 MW) terahertz laser impulses at 5.6 MHz frequency.     

Latest Oligocene through early middle Miocene diatom biostratigraphy of the eastern tropical Pacific

Study of DSDP Sites 71, 77, and 495 has allowed the development of a refined diatom biostratigraphy for the latest Oligocene through early middle Miocene of the eastern tropical Pacific which is well correlated to the low-latitude zonations for planktonic foraminifers, coccoliths, and radiolarians. Six zones and 7 subzones are proposed, and correlation with high-latitude diatoms zonations for the North Pacific, the Norwegian Sea, and the Southern Ocean is suggested by the discovery of selected diatoms in these tropical sediments which were previously thought to be restricted to high latitudes. Six new species and one new variety of diatoms which are stratigraphically useful are proposed: Actinocyclus hajosiae, n. sp., A. radionovae, n. sp., Coscinodiscus blysmos, n. sp., C. praenodulifer, n. sp., Craspedodiscus rydei, n. sp., Thalassiosira bukryi, n. sp., and Coscinodiscus lewisianus var. robustus n. var.     

Trace elements in the otoliths and dorsal spines of albacore tuna (Thunnus alalunga, Bonnaterre, 1788): An assessment of the effectiveness of cleaning procedures at removing postmortem contamination

Trace element analysis or “elemental fingerprinting” is widely used in stock structure analyses. Postmortem contamination of bony structures can confound the results of microconstituent studies or introduce an additional source of noise to the data, thus reducing the ability of the technique to detect real variation in trace element concentrations. Despite the potential for postmortem contamination during sample preparation, the effectiveness of the procedures used to remove potential contaminants from sectioned otoliths and other calcareous structures prior to laser ablation inductively coupled plasma mass spectrometry (LA ICP-MS) has not previously been addressed. Otoliths and dorsal spine sections of albacore tuna (Thunnus alalunga) collected from the North East Atlantic Ocean and the Mediterranean Sea were deliberately contaminated prior to analysis of trace element composition using LA ICP-MS. The effectiveness of three cleaning treatments (rinsing in ultrapure water, 30% hydrogen peroxide and ultrapure 5% nitric acid) at removing this postmortem contamination were compared. Magnesium and strontium were relatively robust to postmortem effects when exposed to contamination at concentrations of 50 ppm and 200 ppm respectively. Soaking in a solution containing Mn, Cs and Ba (50 ppm) caused a marked increase in the detected concentration of each element in both structures. Translucent bands in both structures were more susceptible to contamination. Rinsing in ultrapure water or hydrogen peroxide was not effective at removing Mn, Cs and Ba contamination from either calcareous structure. Washing the otoliths and spines in nitric acid successfully removed postmortem contaminants.The removal of otoliths from tuna damages the appearance of the fish and has an adverse effect on market value. However spines are easily removed, do not affect the appearance or value of the fish and are the most commonly used structure for age determination. A weak but significant correlation was observed between Ba in opaque zones in otoliths and dorsal spines. All other spine to otolith correlations were not significant. The results do not provide support for the use of spines as an alternative to otoliths in trace elemental analyses.     

Cationic amino acids specific biomimetic silicification in ionic liquid: a quest to understand the formation of 3-D structures in diatoms

The intricate, hierarchical, highly reproducible, and exquisite biosilica structures formed by diatoms have generated great interest to understand biosilicification processes in nature. This curiosity is driven by the quest of researchers to understand nature's complexity, which might enable reproducing these elegant natural diatomaceous structures in our laboratories via biomimetics, which is currently beyond the capabilities of material scientists. To this end, significant understanding of the biomolecules involved in biosilicification has been gained, wherein cationic peptides and proteins are found to play a key role in the formation of these exquisite structures. Although biochemical factors responsible for silica formation in diatoms have been studied for decades, the challenge to mimic biosilica structures similar to those synthesized by diatoms in their natural habitats has not hitherto been successful. This has led to an increasingly interesting debate that physico-chemical environment surrounding diatoms might play an additional critical role towards the control of diatom morphologies. The current study demonstrates this proof of concept by using cationic amino acids as catalyst/template/scaffold towards attaining diatom-like silica morphologies under biomimetic conditions in ionic liquids.     

Computational modelling of diatom silicic acid transporters predicts a conserved fold with implications for their function and evolution

Diatoms are an important group of algae that can produce intricate silicified cell walls (frustules). The complex process of silicification involves a set of enigmatic integral membrane proteins that are thought to actively transport the soluble precursor of biosilica, dissolved silicic acid. Full-length silicic acid transporters are found widely across the diatoms while homologous shorter proteins have now been identified in a range of other organisms. It has been suggested that modern silicic acid transporters arose from the union of such partial sequences. Here, we present a computational study of the silicic acid transporters and related transporter-like sequences to help understand the structure, function and evolution of this class of membrane protein. The AlphaFold software predicts that all of the protein sequences studied here share a common fold in the membrane domain which is entirely different from the predicted folds of non-homologous silicic acid transporters from plants. Substrate docking reveals how conserved polar residues could interact with silicic acid at a central solvent-accessible binding site, consistent with an alternating access mechanism of transport. The structural conservation between these proteins supports a model where modern silicon transporters evolved from smaller ancestral proteins by gene fusion.     

The diversity of relationships between Antarctic sponges and diatoms: the case of Mycale acerata Kirkpatrick, 1907 (Porifera,Demospongiae)

The diatom assemblage associated with the Antarctic sponge Mycale acerata was studied through an analysis of the diatom frustule and pigment concentrations in both the sponge ectosome and choanosome. Sponges were sampled weekly from November 2001 to February 2002 at Terra Nova Bay, Antarctica, at a depth of 25–35 m. The most abundant diatoms were Porannulus contentus, Fragilariopsis curta, Thalassiosira cf. gracilis, T. perpusilla and Plagiotropis sp. High abundances of P. contentus were found on the sponge ectosome up to the beginning of November, before the ice melted, while later frustules were incorporated inside, indicating that P. contentus lives epibiontically on M. acerata and represents a potential food source for the sponge. The presence of other diatom species was mainly related to the summer phytoplankton bloom. The sponge incorporates diatoms from the water column and utilises them as a food source, accumulating frustules inside the choanosome. The lack of planktonic diatom frustules at the beginning of the summer indicates that they are expelled or dissolved during the cold season.     

The Diatom Staurosirella pinnata for Photoactive Material Production

A native isolate of the colonial benthic diatom Staurosirella pinnata was cultivated for biosilica production. The silicified cell walls (frustules) were used as a source of homogeneous and structurally predictable porous biosilica for dye trapping and random laser applications. This was coupled with the extraction of lipids from biomass showing potential to fabricate photoactive composite materials sustainably. The strain was selected for its ease of growth in culture and harvesting. Biosilica and lipids were obtained at the end of growth in indoor photobioreactors. Frustules were structurally characterized microscopically and their chemistry analyzed with Fourier Transform Infrared Spectroscopy. Frustule capacity of binding laser dyes was evaluated on a set of frustules/Rhodamine B (Rho B) solutions and with respect to silicon dioxide and diatomite by Fluorescence Spectroscopy demonstrating a high affinity for the organic dye. The effect of dye trapping property in conveying Rho B emission to frustules, with enhancement of scattering events, was analyzed on Rho B doped polyacrylamide gels filled or not with frustules. Amplified spontaneous emission was recorded at increasing pump power indicating the onset of a random laser effect in frustule filled gels at lower power threshold compared to unfilled matrices.     

Morphological investigations of the frustule, perizonium and initial valves of the freshwater diatom Achnanthes crenulata Grunow (Bacillariophyceae)

The present study clarifies the fine structure of the vegetative frustules, initial valves and perizonium of Achnanthes crenulata Grunow. The valves of the vegetative cell are distinctly linear‐lanceolate with an undulate margin. The valve face is quite flat and in girdle view is smoothly curved as in species of Gephyria (Bacillariophyceae). However, the valve face of the initial cells is slightly rounded and does not have an undulate margin. Furthermore, the rapheless sternum is centrally positioned along the apical axis of the araphid initial valve. As this taxon develops from auxospore to initial valve, it forms only longitudinal perizonial bands; no transverse bands arise. The perizonium consists of three silicified bands: one large, central longitudinal plate and two bands that underlie this plate; these two bands are either open or closed. This taxon has several conspicuous structures compared to other marine species of Achnanthes, but the structure of the perizonium supports the position of A. crenulata within Achnanthes sensu stricto.     

Lorica Production in Choanoflagellates–A Model System for Investigating the Mechanics, Controls and Dynamics of Secretion

BIOMINERALIZATION is the process by which living organisms assemble structures from naturally occurring inorganic compounds. Mineral deposition is common and widespread amongst Protozoa and in most instances the mineralized structures provide skeletal support and protection for softer organic parts [10]. The 2 most common minerals to be deposited by Protozoa are silica and calcium carbonate. Groups of Protozoa that deposit silica, which we are concerned with here, include the diatoms, chrysophytes, choanoflagellates, Radiolar-ia, Heliozoa and testate amoebae [10]. In the majority of silica-depositing protista, silica is taken up from the medium in the form of monomelic orthosilicic acid Si(OH)₄ (soluble reactive silicate) and deposited as amorphous, polymerised biogenic silica or opal within membrane-bounded vesicles known as silica deposition vesicles (SDV). Often biogenic silica is characteristically patterned and ornamented and for most protozoan groups the morphology of silicified parts is of prime taxonomic importance. By far the most extensively studied group of silica-depositing organisms are the diatoms [1, 12, 13]. To date most of our knowledge of silica metabolism in protists has been based on investigations into this group. Diatoms require silica for the production of their frustules. Uptake and deposition of silica occurs within a closely denned portion of the cell cycle, between nuclear division and cell separation. It occupies about ± of the cell cycle and without an adequate supply of silica diatoms are unable to produce new frustule valves with the result that cell division cannot be completed. Diatoms, therefore, have an obligate requirement for silica and without this nutrient they cease to grow [11]. In contrast to diatoms a number of other silica-depositing protistan groups, such as loricate choanoflagellates and certain chrysophytes, have a facultative requirement for silica. In the past decade the ultras true ture, physiology and ecology of loricate choanoflagellates have been extensively studied by a number of different workers [7] and the significance of these studies to our understanding of the mechanisms, controls and dynamics of silica secretion is summarised and discussed here.