Biofilm and capsule formation of the diatom Achnanthidium minutissimum are affected by a bacterium

Photoautotrophic biofilms play an important role in various aquatic habitats and are composed of prokaryotic and/or eukaryotic organisms embedded in extracellular polymeric substances (EPS). We have isolated diatoms as well as bacteria from freshwater biofilms to study organismal interactions between representative isolates. We found that bacteria have a strong impact on the biofilm formation of the pennate diatom Achnanthidium minutissimum. This alga produces extracellular capsules of insoluble EPS, mostly carbohydrates (CHO), only in the presence of bacteria (xenic culture). The EPS themselves also have a strong impact on the aggregation and attachment of the algae. In the absence of bacteria (axenic culture), A. minutissimum did not form capsules and the cells grew completely suspended. Fractionation and quantification of CHO revealed that the diatom in axenic culture produces large amounts of soluble CHO, whereas in the xenic culture mainly insoluble CHO were detected. For investigation of biofilm formation by A. minutissimum, a bioassay was established using a diatom satellite Bacteroidetes bacterium that had been shown to induce capsule formation of A. minutissimum. Interestingly, capsule and biofilm induction can be achieved by addition of bacterial spent medium, indicating that soluble hydrophobic molecules produced by the bacterium may mediate the diatom/bacteria interaction. With the designed bioassay, a reliable tool is now available to study the chemical interactions between diatoms and bacteria with consequences for biofilm formation.     

Purification and culture characteristics of 36 benthic marine diatoms isolated from the Solthörn tidal flat (Southern North Sea)

Marine benthic diatoms growing in biofilms on sediment surfaces generally occur associated with heterotrophic bacteria, whereas modern molecular techniques and analyses of species‐specific physiology create a demand for axenic cultures. Numerous benthic diatoms were isolated from surface sediments during a monitoring of the Solthörn tidal flat (southern North Sea, Germany) from May 2008 to May 2009. Of these, around 50% could be purified from the accompanying heterotrophic bacteria using different antibiotics combined with physical separation methods (vortexing, ultrasound). Overall, seven different antibiotics were tested at different concentrations, and a best working protocol was developed. The axenic strains were stable on average for only around 15 months, indicating a symbiotic interaction between the benthic diatoms and the associated bacteria. While most short‐term effects during the purification process were restricted to differences in growth rates among xenic and axenic diatom strains, long‐term cultivation led to distinct changes in cell volumes and growth characteristics of the axenic strains.     

PATTERNS OF CELL SIZE CHANGE IN A MARINE CENTRIC DIATOM: VARIABILITY EVOLVING FROM CLONAL ISOLATES1

Isolates of the centric diatom, Thalassiosira weissflogii Grun., were maintained in exponential growth under constant, favorable conditions for nearly 2 years. During this interval, each culture underwent periodic increases and decreases in mean cell size, a behavior predicted for diatom populations alternating between sexual and asexual reproduction, respectively. The overall patterns of cell size change displayed by each culture, however, were unique. The maximum size of newly enlarged cells varied among isolates and within a given isolate over time. Consequently, both the timing and rate of increase in mean cell size also varied despite the fact that the minimum average cell size obtained by the various cultures was relatively constant. The most consistent feature among the isolates was the rate of decrease in mean cell size, a value determined by the physical constraints of the diatom frustule during mitotic divisions. We hypothesize that the extent of the variability exhibited by these cultures results from the fact that an inherent feature of diatom populations is a constantly changing genetic composition.     

GROWTH OF VITAMIN B12-REQUIRING MARINE DIATOMS IN MIXED LABORATORY CULTURES WITH VITAMIN B12-PRODUCING MARINE BACTERIA12

The vitamin B₁₂ requirement of several marine diatoms can be satisfied in B₁₂^?limited laboratory cultures by heterotrophic marine bacteria isolated from the same waters and from sediments. The bacteria can utilize diatom excretory products, or the remains of dead diatom cells, in the production of the vitamin. The growth of 12 B₁₂¹? requiring diatoms (7 genera) in mixed cultures with 14 different bacteria (without added B₁₂) was compared to the growth of those same diatoms in axenic cultures with excess added B₁₂. Diatom growth was generally rapid in the first few days, followed by sustained, slower growth. The diatom yields in mixed cultures ranged from 0.8 to 84% of the yields in axenic cultures with added B₁₂. In a detailed study of one mixed culture, increases in diatom densities were paralleled by increases in cell densities of the bacterium during the first few days of exponential diatom growth. During the period of slow diatom growth, when diatom densities oscillated but steadily increased, the decreases in diatom densities were associated with increased bacterial growth. This suggests that death of a fraction of the B₁₂-limited diatom population releases sufficient organic matter to stimulate growth of the bacteria and their subsequent excretion of B₁₂; this B₁₂ in turn stimulates further growth of the diatoms. Diatom-bacteria interactions leading to the production of B₁₂ may be important in maintaining viable populations of B₁₂-requiring diatoms in nutrient-poor waters during periods between blooms, when conditions are unfavorable for rapid growth.     

The effect of bacteria on the solubilization of silica in diatom frustules

P atrick , S heila & H olding , A.J. 1985. The effect of bacteria on the solubilization of silica in diatom frustules. Journal of Applied Bacteriology 59 , 7–16.
Natural bacterial populations in samples of water from Loch Leven and Lough Neagh increased the rate of solubilization of frustule silica from an axenic Cyclotella meneghiniana culture, compared with sterile autolysis, at 25C. In the inoculated cultures 50–60% of the silica was solubilized over a period of 30 d. Bacterial populations in Loch Leven water also enhanced the solubilization of silica from non-axenic cultures of Asterionella formosa, Tabellaria flocculosa, Navicula pellicu-losa and C. meneghiniana , compared with control cultures sterilized with mercuric chloride. Similar results were obtained with Lough Neagh populations incubated with A. formosa . In comparison with untreated cells, the treatment of diatom cells with ultra-sonication did not increase the release of silica. Pure cultures of bacteria from Loch Leven water enhanced the release of silica from non-axenic A. formosa and axenic C. meneghiniana compared with sterile control treatments. The variation in the ability of cultures to solubilize the frustule silica appeared to be related to their potential to produce hydrolytic enzymes. Natural populations of Loch Leven and Lough Neagh water bacteria and certain bacterial cultures caused the diatoms to aggregate, which did not enhance the release of silica.     

PROTOCOLS FOR THE REMOVAL OF BACTERIA FROM FRESHWATER BENTHIC DIATOM CULTURES1

In this study, we describe different combinations of physical separation and antibiotic treatment to remove associated bacteria from freshwater diatoms. Diatoms were purified either from natural epilithic biofilms or from unialgal cultures. We determined that for most strains, different purification procedures have to be combined individually. In a new approach, we show that for some diatom strains, the substitution of associated aquatic bacteria by an antibiotic‐sensitive Escherichia coli strain and subsequent treatment with antibiotics may be a successful strategy to obtain axenic diatom cultures. Axenic diatom cultures are essential to study the physiology and biochemistry of individual strains as well as their responses to environmental changes without interference of accompanying bacteria.     

Diversity and distribution of epibiotic bacteria on Pseudo-nitzschia multiseries (Bacillariophyceae) in culture, and comparison with those on diatoms in native seawater

Previous studies on the interaction between bacteria and harmful algal bloom species have mostly considered the bacteria in the bulk solution. Here, we document the abundance and mode of attachment of bacteria growing on the cell surface of the domoic acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle in culture, compared with diatoms in field samples. The epiphytic bacteria were examined by scanning electron microscopy to visualize their morphology and mode of attachment. Two P. multiseries cultures were studied: clone CLN-1 and sub-clone CLN-1-NRC; the latter had been maintained in another laboratory for 2 years. Each of these P. multiseries cultures exhibited a clearly different assemblage of epibiotic bacteria, even though both originated from the same parent culture. The bacterial diversity was greater in clone CLN-1 (nine distinct morphotypes seen) than in sub-clone CLN-1-NRC (six morphotypes). The former clone also produced more domoic acid than the latter. There was a succession of bacterial morphotypes as well as an increase in the number of epiphytic bacteria per diatom cell during the progression from exponential to stationary phase. The most diverse and common morphotypes were rod-shaped cells (e.g. a Caulobacter-like bacterium attached by a discoid holdfast). Epibionts showed a preference for attachment at specific regions of the host diatom frustule, e.g. the raphe or cingulum, locations where organic matter may be extruding from the diatom cell. Most diatom cells carried only one to five bacteria, and up to ca. 60% of the intact diatom cells (although intact cells themselves were infrequent) were still free of epibiotic bacteria at the end of the 31-day batch culture experiment. Sequencing of the SSU rRNA gene showed that five of the eight bacterial strains isolated from the P. multiseries cultures were members of the Alphaproteobacteria, three of the Gammaproteobacteria and one of the Bacteroidetes. A morphologically diverse assemblage of epibiotic bacteria was also found on both centric and pennate planktonic diatoms in natural coastal waters. Of the eight morphotypes recorded, all but two were also found in the cultures. Relatively fewer wild diatom cells carried bacteria compared to cells in culture. We hypothesize that the diversity and abundance of epiphytic bacteria may explain some of the variability seen in the production of DA by different P. multiseries clones, and should be considered as another important and controllable variable that influences diatom cell physiology.     

Radioisotopic Method for Measuring Cell Division Rates of Individual Species of Diatoms from Natural Populations

Silicon is an essential element for diatom frustule synthesis and is usually taken up only by dividing cells. With ⁶⁸Ge, a radioactive analog of Si, the cell cycle marker event of frustule formation was identified for individual species of diatom. The frequency of cells within a population undergoing this division event was estimated, and the cell division rate was calculated. In laboratory cultures, these rates of cell division and those calculated from changes in cell numbers were similar. By dual labeling with ⁶⁸Ge(OH)₄ and NaH¹⁴CO₃, rates of cell division and photosynthesis were coincidently measured for diatoms both in laboratory cultures and when isolated from natural populations in estuarine, offshore, and polar environments. These techniques permit the coupling between photosynthesis and cell division to be examined in situ for individual species of diatom.     

Micro‐photoluminescence of single living diatom cells

Diatoms are single‐celled microalgae that possess a nanostructured, porous biosilica shell called a frustule. This study characterized the micro‐photoluminescence (μ‐PL) emission of single living cells of the photosynthetic marine diatom Thalassiosira pseudonana in response to UV laser irradiation at 325 nm using a confocal Raman microscope. The photoluminescence (PL) spectrum had two primary peaks, one centered at 500–510 nm, which was attributed to the frustule biosilica, and a second peak at 680 nm, which was attributed to auto‐fluorescence of photosynthetic pigments. The portion of the μ‐PL emission spectrum associated with biosilica frustule in the single living diatom cell was similar to that from single biosilica frustules isolated from these diatom cells. The PL emission by the biosilica frustule in the living cell emerged only after cells were cultivated to silicon depletion. The discovery of the discovery of PL emission by the frustule biosilica within a single living diatom itself, not just its isolated frustule, opens up future possibilities for living biosensor applications, where the interaction of diatom cells with other molecules can be probed by μ‐PL spectroscopy. Copyright © 2016 John Wiley & Sons, Ltd.     

Assessing the short-term response of stream diatoms to acidity using inter-basin transplantations and chemical diffusing substrates

1. Acid‐base status has major effects on diatoms, but there is little information on their short‐term response to changing acidity. This is despite the use of diatoms as bioindicators in streams where acid episodes are important during rainstorms (hours to days) or snowmelt (days to weeks). In the Llyn Brianne experimental catchments (Wales, UK), we attempted to mimic the effects of short‐term acidification by (i) reciprocally transplanting diatoms between two streams of contrasting acidity and (ii) using acid‐diffusing substrates. 2. Diatom diversity decreased rapidly on substrata transplanted from the circumneutral into the acidic stream, and increased in the reciprocal transplantation. Changes in dominant taxa occurred within three days in the acidic stream because of the rapid growth of Eunotia exigua, and by nine days in the circumneutral stream because of the proliferation of Achnanthidium minutissimum. Transplants were near indistinguishable from ambient assemblages by day 12. 3. There were no effects of enclosures on assemblage composition, but diatoms responded more rapidly to altered chemistry in enclosures with coarse mesh (26 × 50 mm) than finer mesh (320 μm). 4. Chemical diffusing substrates comprised terracotta tiles attached to dosing reservoirs that created locally acid (using H₂SO₄) or metal‐rich conditions (using MnSO₄) in the circumneutral stream over 26 days. Diatom responses were compared with reference substrates dosed with deionised or circumneutral stream water, and we also assessed whether effects were moderated by macroinvertebrate grazers. 5. Surface pH was lower by 1–2 pH units on acid‐dosed substrates than on reference tiles or in surrounding streamwater. Grazed assemblages on acid‐dosed substrates differed significantly from ungrazed reference assemblages, acquiring significantly greater relative abundance of Eunotia spp. However, the magnitude of response was less than in the between‐stream transplantations either because (i) metal exposure and base cation concentrations differed between the transplants and dosing substrates or (ii) diatom response to reduced pH on the diffusing substrates was restricted by the scarcity of acidobiontic diatoms in the circumneutral stream. Similar filter, founder or dominance effects might also affect diatom responses to real acid episodes. 6. These data show that diatom assemblages can respond rapidly and directly to changes in acid‐base status, but short‐term acidification might affect diatoms more rapidly than subsequent recovery. Because the experimental methods used were imperfect representations of episodic effects, diatom response to real acid events requires further field evaluation.     

Allelopathic effects of Alexandrium fundyense (Dinophyceae) on Thalassiosira cf. gravida (Bacillariophyceae): a matter of size

Allelopathic interactions among phytoplankton are well documented. The potency of allelopathic species and responses of target species to allelochemicals are quite variable, however, limiting full understanding of the role these interactions may play in nature. One trait that may influence the sensitivity of an individual to allelochemicals is cell size. The few studies that have examined relationships between cell size and susceptibility to allelochemicals have compared different species and thus could not distinguish between the role of size and species‐specific physiological differences. Culturing an actively sexually reproducing diatom allowed us to focus on the influence of target cell size within a single species. We studied growth and nutrient acquisition by the chain‐forming Thalassiosira cf. gravida Clever in the presence and absence of allelochemicals released by Alexandrium fundyense Balech as a function of T. cf. gravida cell size. Upon exposure to filtrate of A. fundyense, T. cf. gravida cultures “bleached” and both growth and nutrient utilization ceased for up to 4 d. The magnitude of the effect was dependent on filtrate concentration and T. cf. gravida cell surface area:volume ratio. The greatest inhibition was observed on the smallest cells, while T. cf. gravida cultures that had undergone cell enlargement via sexual reproduction were least sensitive to A. fundyense filtrate. These results demonstrate that competitor cell size, independent from taxonomy, may influence the outcome of allelopathic interactions. The findings presented here suggest a potential ecological impact of diatom cell size reduction and sexual reproduction that has not yet been described and that may be important in determining diatom survival and success.     

Influence of bacteria and salinity on diatom biogenic silica dissolution in estuarine systems

Dissolution of diatom biogenic silica (bSiO₂) in estuaries and its control by water salinity and bacteria were investigated using the river euryhaline species Cyclotella meneghiniana as a model. Laboratory-controlled bioassays conducted at different salinities with an estuarine bacteria inoculum showed a faster dissolution of diatom bSiO₂ at the lowest salinity where bacteria were the most abundant. However in another experiment, salinity increase clearly enhanced the dissolution of cleaned frustules (organic matter free). The presence of active bacteria might therefore predominate on the effect of salinity for freshly lysed diatoms whereas salinity might rather control dissolution of organic-matter-free frustule remains. Incubation of cultivated diatoms at different protease concentrations revealed that high proteolytic activities had little effect on bSiO₂ dissolution at a 1-month scale in spite of an efficient removal of organic matter from the frustules. Altogether it is hypothesized that bacterial colonization increases bSiO₂ dissolution by creating a microenvironment at the diatom surface with high ectoproteolytic activity but also via the release of metabolic byproducts since the presence of organic matter seems generally to facilitate diatom bSiO₂ dissolution.     

MORPHOLOGICAL FLEXIBILITY OF COCCONEIS PLACENTULA (BACILLARIOPHYCEAE) NANOSTRUCTURE TO CHANGING SALINITY LEVELS1

Diatoms possess a silica frustule decorated with unique patterns of nanosize features. Here, we show for the first time from in situ samples that the size of the nanopores present at the surface of the diatom Cocconeis placentula Ehrenb. varies with fluctuating salinity levels. The observed reduction in nanopore size with decreasing salinity agrees with previous laboratory experiments. We also uniquely combined our observations with theoretical considerations to demonstrate that the decrease in the diffusive layer thickness is compensated for by the changes in pore size, which maintain a steady diffusive flux toward the diatom’s cell at different salinities. This process allows diatoms to absorb similar amount of nutrients whatever the salinity and as such to increase their ecological competitiveness in fluctuating environments. These results further suggest that the overall ecological success of diatoms, and their ability to react to environmental changes, may be controlled by the flexibility of the morphological characteristics of their frustules.     

SEXUALITY, INCOMPATIBILITY, SIZE VARIATION, AND PREFERENTIAL POLYANDRY IN NATURAL POPULATIONS AND CLONES OF SELLAPHORA PUPULA (BACILLARIOPHYCEAE)

The capitate and rectangular demes of the freshwater epipelic diatom Sellaphora pupula (Kütz.) Mereschk. are dioecious, the first such report for any freshwater diatom. Sexual differentiation, which is probably determined genetically, involves recognition at the cell surface as well as differences in gamete behavior (one gametangium produces an active "male" gamete, the other a passive "female" gamete). In culture, successful sexual reproduction occurs only when compatible clones are mixed. All cells of a clone behave identically in interclonal crosses, being either male or female, regardless of the stage of the life cycle, in contrast to the sequential hermaphroditism of centric diatoms. Males and females have identical frustule morphology. As in other diatoms, there is an upper size threshold for sexual reproduction, below which cells become progressively easier to sexualize. In culture, sexual interactions occur in cells much smaller than those ever seen in natural populations, so that in nature the sexual size range is effectively open. Natural populations almost always contain sexualizable cells; often, most of the cells are below the upper sexual size threshold. Male gametangia are, on average, slightly larger than females in the capitate deme, which may be produced by preferential polyandry, depleting the population of males and making them younger at mating. Rarely, selfing occurs producing zygotes, but these abort before producing initial cells. The sizes of the gametangia and initial cells are correlated but this does not invalidate the use of "cardinal points" of the life cycle in taxonomy. No interbreeding occurs between the rectangular and capitate demes. However, when males of one deme are mixed with females of the other, there is a stimulation of activity, as during the early stages of pairing in compatible intrademic crosses.     

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans

Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and >200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion. Received: 2 December 1996 / Accepted: 6 March 1997     

NANOSTRUCTURE OF THE DIATOM FRUSTULE AS REVEALED BY ATOMIC FORCE AND SCANNING ELECTRON MICROSCOPY

The cell wall (frustule) of the freshwater diatom Pinnularia viridis (Nitzsch) Ehrenberg is composed of an assembly of highly silicified components and associated organic layers. We used atomic force microscopy (AFM) to investigate the nanostructure and relationship between the outermost surface organics and the siliceous frustule components of live diatoms under natural hydrated conditions. Contact mode AFM imaging revealed that the walls were coated in a thick mucilaginous material that was interrupted only in the vicinity of the raphe fissure. Analysis of this mucilage by force mode AFM demonstrated it to be a nonadhesive, soft, and compressible material. Application of greater force to the sample during repeated scanning enabled the mucilage to be swept from the hard underlying siliceous components and piled into columns on either side of the scan area by the scanning action of the tip. The mucilage columns remained intact for several hours without dissolving or settling back onto the cleaned valve surface, thereby revealing a cohesiveness that suggested a degree of cross-linking. The hard silicified surfaces of the diatom frustule appeared to be relatively smooth when living cells were imaged by AFM or when field-emission SEM was used to image chemically cleaned walls. AFM analysis of P. viridis frustules cleaved in cross-section revealed the nanostructure of the valve silica to be composed of a conglomerate of packed silica spheres that were 44.8 ± 0.7 nm in diameter. The silica spheres that comprised the girdle band biosilica were 40.3 ± 0.8 nm in diameter. Analysis of another heavily silicified diatom, Hantzschia amphioxys (Ehrenberg) Grunow, showed that the valve biosilica was composed of packed silica spheres that were 37.1 ± 1.4 nm and that silica particles from the girdle bands were 38.1 ± 0.5 nm. These results showed little variation in the size range of the silica particles within a particular frustule component (valve or girdle band), but there may be differences in particle size between these components within a diatom frustule and significant differences are found between species.     

GROWTH INHIBITION AND TOXICITY OF THE DIATOM ALDEHYDE 2‐TRANS, 4‐TRANS‐DECADIENAL ON THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

A common aldehyde present in marine and freshwater diatoms, 2‐trans, 4‐trans‐decadienal (A3), is involved in the wound‐activated response of diatoms to copepod grazing. Upon breakage of the diatom cell membrane, aldehydes are enzymatically produced by the rapid conversion of precursors and strongly impact copepod reproduction by impairing egg production and hatching success, inducing teratogenic embryos modifications. In this study, A3 was assayed with the marine diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle. The aldehyde concentration necessary to reduce 50% growth rate (EC₅₀) was 0.29 mg·L^?1. Decadienal was found to inhibit T. weissflogii cell growth in a dose‐ and time‐dependent manner, with irreversible effects after 24 h of exposure. Decadienal induced a degenerative process, through modifications of cell membrane characteristics, interference with cell cycle progression, and with cell metabolic activity, leading to cell death. A preferential action of A3 on dividing cells was observed. Photosynthetic efficiency significantly decreased upon exposure to the aldehyde, paralleled by an increase in diatoxanthin, suggesting a protective role of this xanthophyll, usually involved in photoprotection. Dying cells exhibited the morphological and biochemical features that bear close resemblance to apoptosis of mammalian cells, including cell shrinkage, chromatin condensation, and degradation of nuclear DNA to nucleosomal size fragments. These data are the first direct evidence to show aldehydes are toxic to diatoms. We suggest a possible nontoxic role of such compounds as chemical signals of unfavorable conditions within the phytoplankton communities, which may be relevant for the population dynamics of diatoms during blooms.