Iron incorporation in biosilica of the marine diatom <Emphasis Type="Italic">Stephanopyxis</Emphasis> <Emphasis Type="Italic">turris</Emphasis>: dispersed or clustered?

Iron incorporation into diatom biosilica was investigated for the species Stephanopyxis turris. It is known that several “foreign” elements (e.g., germanium, titanium, aluminum, zinc, iron) can be incorporated into the siliceous cell walls of diatoms in addition to silicon dioxide (SiO₂). In order to examine the amount and form of iron incorporation, the iron content in the growth medium was varied during cultivation. Fe:Si ratios of isolated cell walls were measured by ICP-OES. SEM studies were performed to examine of a possible influence of excess iron during diatom growth upon cell wall formation. The chemical state of biosilica-attached iron was characterized by a combination of infrared, ²⁹Si MAS NMR, and EPR spectroscopy. For comparison, synthetic silicagels of variable iron content were studied. Our investigations show that iron incorporation in biosilica is limited. More than 95% of biosilica-attached iron is found in the form of iron clusters/nanoparticles. In contrast, iron is preferentially dispersedly incorporated within the silica framework in synthetic silicagels leading to Si–O–Fe bond formation.     

Media optimization and lipid formation of two native diatoms for cultivation in the Southwest Texas desert

Two native diatom isolates belonging to the genus Nitzschia, TAMU-LBK-020 and TAMU-LBK-023, were grown in a chamber simulating summer temperatures and light:dark cycle in the southwest Texas (USA) desert. Different concentrations of macronutrients (Si, N, and P) were tested to identify conditions conducive to growth. Growth rates of the fastest growing treatments exceeded 0.6 doublings per day, with DW and AFDW exceeding 1.0 and 0.8 g L^?1, respectively. Treatments with positive growth were continued to a lipid formation step by omission of Si from the medium in this phase, with lipid accumulation monitored daily by Nile red fluorescence. Peak oil was observed at approximately day 3 for TAMU-LBK-020 and day 5 for TAMU-LBK-023. The highest oil concentrations observed were 17.5?±?7.3 % of AFDW in TAMU-LBK-020 and 28.5?±?5.6 % of AFDW in TAMU-LBK-023 at low N concentrations, but the low N also resulted in growth of 0.2 to 0.5 doublings per day. We propose a biphasic growth strategy, initially under a high macronutrient concentration to promote cell growth and division followed by starving for Si and N to achieve maximal lipid production. However, we anticipate that it will be necessary to determine optimal nutrient concentrations empirically for each species and strain. These results demonstrate that diatoms used for biofuels can achieve fast growth rates and high oil production, and also that native strains, when properly cultured, can be highly suitable for biofuel production, even in harsh climates such as the desert Southwest U.S.     

Aluminium speciation in the presence of wheat root cell walls: a wet chemical study

Hydrolysis of Al³⁺ was performed in the presence of isolated root cell walls from a series of wheat cultivars (Triticum aestivum L.) known to have differential tolerance to Al contamination. Aluminium speciation was dependent on the cell wall concentration. At low cell wall concentrations, significant amounts of the very toxic Al₁₃ species were formed. At higher cell wall concentrations, formation of the tridecamer was hindered or completely inhibited. The sensitive wheat cultivars displayed a higher affinity for aluminium than the tolerant cultivars. A possible Al tolerance mechanism based on cell wall permeability is discussed.     

Diatom elemental and morphological changes in response to iron limitation: a brief review with potential paleoceanographic applications

Diatoms are a major group of phytoplankton that account for approximately 40% of the ocean carbon fixation and the vast majority of biogenic silica production through the construction of their cell walls (termed frustules). These frustules accumulate and are partially preserved in the ocean sediments. Diatom growth and nutrient utilization in high‐nitrate, low‐chlorophyll regions of the world’s oceans are mostly regulated by iron availability. Diatoms acclimate to iron limitation by decreasing cell size. The associated increase in surface area‐to‐volume ratio and decrease in diffusive boundary layer thickness may improve nutrient uptake kinetics. In parallel, cellular silicon (Si) contents are elevated in iron‐limited diatoms relative to nitrogen (N) and carbon (C). Variations in degree of silicification and nutritional requirements of iron‐limited diatoms have been hypothesized to account for higher cellular Si and/or lower cellular N and C, respectively. However, in some diatoms, frustule silicification does not significantly change when cells are iron‐limited. Instead, changes in the Si‐containing valve surface area relative to volume within these diatoms is hypothesized to be responsible for the variations in the cellular Si : N and Si : C ratios. In particular, some examined iron‐limited pennate diatoms have reduced widths relative to their lengths (i.e. lower length‐normalized widths, LNW) compared to iron‐replete cells. In the pennate diatom Fragilariopsis kerguelensis, the mean LNWs of valves preserved in sediments throughout the Southern Ocean (a well‐characterized iron‐limited region) is positively correlated with satellite‐derived, climatological net primary productivity in the overlying waters. Because of the specific morphological changes in pennate diatom frustules in response to iron availability, the valve morphometerics (e.g. LNWs) can potentially be used as a diagnostic tool for iron‐limited diatom growth and relative changes in the Si : N (and Si : C) ratios in extant diatom assemblages as well as those preserved in the sediments.     

Small angle neutron scattering on an absolute intensity scale and the internal surface of diatom frustules from three species of differing morphologies

The internal nanostructure of the diatoms Cyclotella meneghiniana, Seminavis robusta and Achnanthes subsessilis was investigated using small angle neutron scattering (SANS) to examine thin biosilica samples, consisting of isotropic (powder) from their isolated cell walls. The interpretation of SANS data was assisted by several other measurements. The N₂ adsorption, interpreted within the Branuer–Emmet–Teller isotherm, yielded the specific surface area of the material. Fourier transform infrared (FTIR) and Raman spectroscopy indicates that the isolated material is amorphous silica with small amounts of organic cell wall materials acting as a filling material between the silica particles. A two-phase (air and amorphous silica) model was used to interpret small angle neutron scattering data. After correction for instrumental resolution, the measurements on two SANS instruments covered an extended range of scattering vectors 0.0011 nm^?1 < q < 5.6 nm^?1, giving an almost continuous SANS curve over a range of scattering vectors, q, on an absolute scale of intensity for each sample. Each of the samples gave a characteristic scattering curve where log (intensity) versus log (q) has a ?4 dependence, with other features superimposed. In the high-q regime, departure from this behaviour was observed at a length-scales equivalent to the proposed unitary silica particle. The limiting Porod scattering law was used to determine the specific area per unit of volume of each sample illuminated by the neutron beam. The Porod behaviour, and divergence from this behaviour, is discussed in terms of various structural features and the proposed mechanisms for the bio-assembly of unitary silica particles in frustules.     

Culture and Motion Analysis of Diatom Bacillaria paradoxa on a Microfluidic Platform

We proved the feasibility of using a microfluidic chip to culture diatom Bacillaria paradoxa, and analyzed the gliding characteristics of its self-organized colony in detail. The optimal cultivation parameters of B. paradoxa for the designed chip made with polydimethylsiloxane are as follows: the preferable cells injecting rate for keeping the cells alive is 0.2 mL/h, the initial cell density for fast reproduction is 5.5 × 10⁴ cells/mL, and the optimal replacement period of culture medium is 4 days. B. paradoxa tends to form a colony during their growth, and the colony can glide with a steady period of 29 ± 3 s along its axial direction in a constant stream, the amplitude of the colony will not decay (e.g., 24 μm of two-cell colony at 1.1 mm/s flow rate), and the colony rapidly adjusts its direction of gliding to the direction of water flow. The successful culture of diatoms on a microfluidic platform may be used for biosensing chips and the creation of gasoline-producing diatom solar panels.     

The paths of Andrew A. Benson: a radio-autobiography

Among marine phytoplankton groups, diatoms span the widest range of cell size, with resulting effects upon their nitrogen uptake, photosynthesis and growth responses to light. We grew two strains of marine centric diatoms differing by ~4 orders of magnitude in cell biovolume in high (enriched artificial seawater with ~500 µmol L^?1 µmol L^?1 NO₃ ^?) and lower-nitrogen (enriched artificial seawater with <10 µmol L^?1 NO₃ ^?) media, across a range of growth light levels. Nitrogen and total protein per cell decreased with increasing growth light in both species when grown under the lower-nitrogen media. Cells growing under lower-nitrogen media increased their cellular allocation to RUBISCO and their rate of electron transport away from PSII, for the smaller diatom under low growth light and for the larger diatom across the range of growth lights. The smaller coastal diatom Thalassiosira pseudonana is able to exploit high nitrogen in growth media by up-regulating growth rate, but the same high-nitrogen growth media inhibits growth of the larger diatom species.     

EFFECTS OF Si:P CONCENTRATION RATIOS AND NUTRIENT LIMITATION ON THE CELLULAR COMPOSITION AND MORPHOLOGY OF ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE)1

The freshwater diatom Asterionella formosa Haas. was grown in semicontinuous culture at 20°C under continuous cool-white fluorescent light of ca. 20 μEin · m^?2· s ·^?1 in a medium containing Si: P in various concentration ratios. The cell quotas of P and Si changed in relation to the available concentrations of P and Si at constant μ= 0.11 and 0.16 d^?1. Under Si-limitation, the P cell quota increased by over an order of magnitude as the influent [Si:P] decreased. The Si cell quota increased with increase in [Si] in the influent medium, and it increased as [P] increased at a specific [Si]. Under P-limitation, the P cell quotas were fairly constant and low; the Si cell quotas were relatively high and decreased slightly as influent [P] and [Si] increased. Asterionella stored up to 28 times more P and 2 times more Si than needed. The number of Asterionella cells per colony varied as a function of the influent [Si:P] and nutrient limitation being usually less than or equal to 6 when P-limited, and greater than 10 when Si-limited.     

Induction of Protease Release of the Resistant Diatom Chaetoceros didymus in Response to Lytic Enzymes from an Algicidal Bacterium

Marine lytic bacteria can have a substantial effect on phytoplankton and are even capable to terminate blooms of microalgae. The bacterium Kordia algicida was reported to lyse cells of the diatom Skeletonema costatum and several other diatoms by a quorum sensing controlled excretion of proteases. However the diatom Chaetoceros didymus is fully resistant against the bacterial enzymes. We show that the growth curve of this diatom is essentially unaffected by addition of bacterial filtrates that are active against other diatoms. By monitoring proteases from the medium using zymography and fluorescence based activity assays we demonstrate that C. didymus responds to the presence of the lytic bacteria with the induced production of algal proteases. These proteases exhibit a substantially increased activity compared to the bacterial counterparts. The induction is also triggered by signals in the supernatant of a K. algicida culture. Size fractionation shows that only the >30 kD fraction of the bacterial exudates acts as an inducing cue. Implications for a potential induced defense of the diatom C. didymus are discussed.     

Modelling Si-N-limited growth of diatoms

A mechanistic model for silicon (Si) physiology is developed,interfaced with a model of nitrogen (N) physiology, which iscapable of simulating the major documented facets of Si–Nphysiology in diatoms. The model contains a cell cycle componentthat is involved in regulating the timing of the synthesis ofvalves, girdles and setae. In addition to reproducing the timingof diatom cell division within a light–dark cycle, themodel simulates the following features seen in real diatoms.Synthesis of valves only occurs during G₂ interphase and M,while the girdles and (if appropriate) setae are synthesizedduring G₁. Si stress alone results in a loss of setae, followedby a thinning of the valves in successive generations untila minimum Si cell quota is attained. After this point, the durationof G₂ increases and growth is Si limited. Concurrently, thecarbon (C) cell quota increases, offering the capability tosimulate the documented increase in sinking rates with Si stress.N stress alone results in an increase in the duration of G₁and G₂ interphases, and high Si cell quotas. From this complexmodel, which must be run for arrays of subpopulations to simulatenon-synchronous growth, a simpler model is developed. This iscapable of reproducing similar growth dynamics, although withno reference to component parts of the frustule. When alliedto a photoacclimative submodel, a prediction of the model isthat diatoms starved of Si will release increased amounts ofdissolved organic C because cell growth is halted more rapidlythan the photosystems can be degraded.     

Growth of the plankton diatom Thalassiosira nordenskioeldii Cleve at low silicate concentrations

The combined effect of low silicate concentration and temperature on the growth of the marine plankton diatom Thalassiosira nordenskioeldii Cleve was investigated by means of batch and semi-continuous cultures. Growth rates were measured in thin cell suspensions (less than 500 cells/ml) to prevent the silicate concentration in the medium from decreasing by more than 25 % during the period of measurement.Half-saturation constants of silicate-limited growth were calculated according to the Michaelis-Menten equation. At 3 °C, the constant was 0.09 μg-at. Si/I and at 10 °C, 0.02μg-at. Si/I. In semicontinuous cultures grown for one month at silicate concentrations of 0.3-0.4μg-at. Si/I, the mean cell division rates were 80–100% of the maximum rates recorded at the respective temperatures.It seems unlikely that decreasing silicate concentrations could influence the course of the spring succession of plankton diatom species in arctic or temperate coastal waters.     

Accumulation of aluminium in the cell wall pectin in cultured tobacco (Nicotiana tabacum L.) cells treated with a combination of aluminium and iron

In nutrient medium, aluminium (Al) accumulation in tobacco cells occurs only in the presence of ferrous ion [Fe(II)]. The localization of Al was examined to elucidate a mechanism of Al accumulation. After the digestion of Al-treated cells with cellulase and pectolyase together, the resulting spheroplasts contained as much Al as the intact cells. However, the cell walls isolated from Al-treated cells also contained as much Al as the intact cells. Comparison of sugar and Al contents in polysaccharide components extracted chemically from cell walls isolated from intact cells and spheroplasts revealed that the enzymes digested most of the cellulose and hemicellulose, but only half of the pectin, and that Al mainly existed in the pectin remaining in the spheroplasts. Gel-permeation chromatography of the pectin fraction (NH₄-oxalate extract) from the cell walls of the intact cells indicated that Al was associated with small polysaccharides of approximately 3–7 kDa. These results suggest that a minor part of pectin is a major site of Al accumulation. The content of cell wall pectin increased during Al treatment in nutrient medium. Taken together, we hypothesize that Al may bind to the pectin newly produced during Al treatment.     

Effects of nitrogen delivery on chitin nanofiber production during batch cultivation of the diatom <Emphasis Type="Italic">Cyclotella</Emphasis> sp. in a bubble column photobioreactor

The photosynthetic diatom Cyclotella sp. extrudes chitin nanofibers following cell division. This diatom requires silicon for cell wall biosynthesis and division, as well as nitrogen for biosynthesis of intracellular material and extracellular chitin, an N-acetyl glucosamine biopolymer. The initial nitrogen/silicon molar ratio was the critical parameter for assessing the limits of nitrogen delivery on cell number and chitin production during batch cultivation of Cyclotella in a bubble column photobioreactor under silicon-limited growth conditions, using nitrate as the nitrogen source. The peak rate of volumetric chitin production increased linearly, from 3.0 to 46 mg chitin L^?1 day^?1, with increasing N/Si ratio over the range studied (0.82 to 8.6 mol N mol^?1 Si). However, the cell number yield and the chitin yield per cell increased asymptotically with increasing N/Si ratio, achieving a final cell number yield of 5.3?×?10⁹?±?2.6?×?10⁸ cells mol^?1 Si and chitin yield of 28.7?±?1.2 mg chitin per 10⁹ cells (1.0 S.E.). An N/Si ratio of at least 4.0 mol N mol^?1 Si achieved 90% of the asymptotic chitin yield. This study has shown that scalable cultivation systems for maximizing chitin nanofiber production will require delivery of both silicon and optimal nitrogen under silicon-limiting growth conditions to promote cell division and subsequent chitin formation.     

Effects of manganese on the viability of vegetative diaspores of the epiphytic lichen Hypogymnia physodes

Soredia of the lichen Hypogymnia physodes cultivated with Bold's basal medium on agar plates for 8 days exhibited decreasing growth rates along with increasing Mn concentrations above 3 mM. Ca and Mg added separately or in combination, alleviated Mn toxicity. The chlorophyll a and b content of the soredia was reduced under the influence of Mn and was positively correlated with the rate of grown soredia. Trebouxia cells of the soredia grown with excess Mn were smaller than control cells, had reduced chloroplasts and were partly collapsed; fungal hyphae were shortened and strongly swollen. Disintegrated cell walls occurred both in algal and fungal cells. Excess Mn was sequestered in extracellular encrustations together with phosphate as corresponding anion. Intracellularly, Mn was accumulated in polyphosphate granules both in algal and fungal cells. Mn uptake was correlated with significant loss of Na, Mg and Ca, particularly from the mycobiont. Fungal cell walls also lost significant amounts of P. The same damage symptoms occurred in cells of soredia both grown or not, but the former had a higher share of intact cells. Damaged cells of both types of soredia had equally increased Mn concentrations, whereas the total Mn content was higher in not grown soredia than in the grown ones due to the greater amount of damaged cells in the former. The Si–Mn ratio in cell walls of intact Trebouxia cells was significantly higher than in collapsed cells. The experimental evidence of Mn sensitivity of H. physodes soredia corresponds to studies of epiphyte vegetation in montane spruce forests of northern Germany that revealed decreasing cover values of H. physodes with an increasing Mn content of the substrate.     

The kinetics of aluminum adsorption and desorption by root cell walls of an aluminum resistant wheat (Triticum aestivum L.) cultivar

In the present study, root cell walls were extracted from an Al-resistant wheat (Triticum aestivum L.) cultivar, Atlas 66 to investigate the effect of cell-wall properties on the kinetics of Al adsorption and desorption. Nearly all the Al adsorbed was desorbed by 2.5 mM CaCl₂ at pH 4.5, indicating that most of Al ions were electrically bound to cell wall materials. After the cell walls were treated with 1% pectinase for 30 min to degrade part of pectin, the total amount of Al absorbed was decreased by about 50%, indicating that pectin in the cell walls played an important role in binding Al. When the cell walls were preincubated in 1 and 10 mM malate solution overnight to mimic the organic acid secretion by the roots of wheat, the total amount of Al adsorbed was decreased by 60 and 80%, respectively, suggesting that the malate secreted in response to Al stress not only detoxifies Al by its chelating effect, but also reduce the cell walls' capacity to bind Al.     

Phytoplankton community composition and photosynthetic physiology in the Australian sector of the Southern Ocean during the austral summer of 2010/2011

Phytoplankton population dynamics play an important role in biogeochemical cycles in the Southern Ocean during austral summer. However, the relationship between phytoplankton community composition and primary productivity remains elusive in this region. We investigated the community composition and photosynthetic physiology of surface phytoplankton assemblages in the Australian sector of the Southern Ocean from December 2010 to January 2011. There were significant latitudinal variations in hydrographic and biological parameters along 110°E and 140°E. Surface (5 m) chlorophyll a (chl a) concentrations measured with high-performance liquid chromatography varied between 0.18 and 0.99 mg m^?3. The diatom contribution to the surface chl a biomass increased in the south, as estimated with algal chemotaxonomic pigment markers, while the contributions of haptophytes and chlorophytes decreased. In our photosynthesis–irradiance (P–E) curve experiment, the maximum photosynthetic rate normalized to chl a ( \(P_{ \hbox{max} }^{*}\) ), initial slope (α ^*), the maximum quantum yield of carbon fixation (Φ _{c max}), and the photoinhibition index (β ^*) were higher in the region where diatoms contributed >50 % to the chl a biomass. In addition, there were statistically significant correlations between the diatom contribution to the chl a biomass and the P–E parameters. These results suggested that the changes in the phytoplankton community composition, primarily in diatoms, could strongly affect photosynthetic physiology in the Australian sector of the Southern Ocean.     

Influence of silicon on cobalt, zinc, and magnesium in baker's yeast, Saccharomyces cerevisiae

Silicon (Si, as silicate) is involved in numerous important structure and function roles in a wide range of organisms, including man. Silicate availability influences metal concentrations within various cell and tissue types, but, as yet, clear mechanisms for such an influence have been discovered only within the diatoms and sponges. In this study, the influence of silicate on the intracellular accumulation of metals was investigated in baker's yeast (Saccharomyces cerevisiae). It was found that at concentrations up to 10 mM, silicate did not influence the growth rate of S. cerevisiae within a standard complete medium. However, an 11% growth inhibition was observed when silicate was present at 100 mM. Intracellular metal concentrations were investigated in yeast cultures grown without added silicate (−Si) or with the addition of 10 mM silicate (+Si). Decreased amounts of Co (52%), Mn (35%), and Fe (20%) were found within +Si-grown yeast cultures as compared to −Si-grown ones, whereas increased amounts of Mo (56%) and Mg (38%) were found. The amounts of Zn and K were apparently unaffected by the presence of silicon. +Si enhanced the yeast growth rate for low-Zn²⁺ medium, but it decreased the growth rate under conditions of a low Mg²⁺ medium and did not alter the growth rates in high Zn²⁺ and Co²⁺ media. +Si doubled the uptake rate of Co²⁺ but did not influence that of Zn²⁺. We propose that a possible explanation for these results is that polysilicate formation at the cell wall changes the cell wall binding capacity for metal ions. The toxicity of silicate was compared to germanium (Ge, as GeO₂), a member of the same group of elements as Si (group 14). Hence, Si and Ge are chemically similar, but silicate starts to polymerize to oligomers above 5 mM, whereas Ge salts remain as monomers at such concentrations. Ge proved to be far more toxic to yeast than Si and no influence of Si on Ge toxicity was found. We propose that these results relate to differences in cellular uptake.     

Development of molecular tools for the detection of freshwater diatoms

This study was undertaken to develop a reliable and reproducible procedure for the detection and quantitative determination of diatoms in environmental samples. A comparative study of seven different DNA extraction kits was carried out to establish conditions for analysis of diatom containing samples. The best performers were identified using both standard and real-time PCR. We show that the yield of diatom DNA is generally quite low when using commercially available extraction kits; in addition, a new protocol was devised to obtain samples suitable for DNA amplification without the need to perform all the steps required for DNA extraction. This method was tested on environmental samples spiked, in a wide range of total cell mass, with the rarely occurring diatom Neidium affine together with a highly species-specific oligonucleotide designed on the small subunit (SSU) rRNA gene. Thus, we propose a fast and effective procedure that, combined with the use of species-specific oligonucleotide probes can detect minute amounts of a spiked diatom within a complex diatom community. This study provides experimental conditions for a fast and accurate detection of diatoms, and demonstrates the feasibility of the use of molecular tools in the evaluation of water quality.     

New insights into the complex architecture of siliceous copepod teeth

Copepods belong to the dominant marine zooplankton taxa and play an important role in particle and energy fluxes of the marine water column. Their mandibular gnathobases possess tooth-like structures, so-called teeth. In species feeding on large proportions of diatoms these teeth often contain silica, which is very probably the result of a coevolution with the siliceous diatom frustules. Detailed knowledge of the morphology and composition of the siliceous teeth is essential for understanding their functioning and their significance in the context of feeding interactions between copepods and diatoms. Based on analyses of the gnathobases of the Antarctic copepod Rhincalanus gigas, the present study clearly shows, for the first time, that the silica in the siliceous teeth features large proportions of crystalline silica that is consistent with the mineral α-cristobalite and is doped with aluminium. The siliceous structures have internal chitinous fibre networks, which are assumed to serve as scaffolds during the silicification process. The compact siliceous teeth of R. gigas are accompanied by structures with large proportions of the elastic protein resilin, likely reducing the mechanical damage of the teeth when the copepods feed on diatoms with very stable frustules. The results indicate that the coevolution with diatom frustules has resulted in gnathobases exhibiting highly sophisticated composite structures.