Chronicle of Marine Diatom Culturing Techniques

Diatoms are regarded as useful neutral lipid sources, as liquid fuel precursors, as foods for marine culture of zooplankters, larval and post-larval shrimp, copepods, juvenile oysters and as micromachines in nanotechnology. Combining microscopic observation with in situ culturing has been useful in areas of taxonomy, ecology, biomonitoring, biotechnology, etc. This communication reviews various culturing techniques of marine diatoms with the relative merits.

Mass culture of marine diatom Skeletonema costatum (Greville) Cleve collected from the Bay of Bengal

The growth of Skeletonema costatum in two artificial nutrient media was studied using various culture vessels. Skeletonema costatum was collected from the Cox's Bazar coast around the Bay of Bengal. Different growths stages i.e. lag phase, exponential phase, prestationary phase, stationary and death phase were observed during the culture period. The number of cells increased during the active division period and decreased after the beginning of the prestationary phase. The average densities of S. costatum in primary and secondary cultures were 0.55 x 10 super(6) cells mlˉ¹ and 0.93x10 super(6) cells mlˉ¹, respectively. In mass culture of S. costatum two, types of media were used. Highest cells densities of S. costatum cement tank culture were recorded 1.23x10 super(6) cell mlˉ¹ and 0.78x10 super(6) cells mlˉ¹ in their respective f/4 medium and commercial fertilizer medium. In the cement tanks culture fertilizer medium was found to be the best medium for mass culture of S. costatum in respect of production efficiency and culture stability.

Light histories influence the impacts of solar ultraviolet radiation on photosynthesis and growth in a marine diatom, Skeletonema costatum

Phytoplanktonic species acclimated to high light are known to show less photoinhibition. However, little has been documented on how cells grown under indoor conditions for decades without exposure to UV radiation (UVR, 280-400 nm) would respond differently to solar UVR compared to those in situ grown under natural solar radiation. Here, we have shown the comparative photosynthetic and growth responses to solar UVR in an indoor-(IS) and a naturally grown (WS) Skeletonema costatum type. In short-term experiment (<1 day), phi(PSII) and photosynthetic carbon fixation rate were more inhibited by UVR in the IS than in the WS cells. The rate of UVR-induced damages of PSII was faster and their repair was significantly slower in IS than in WS. Even under changing solar radiation simulated for vertical mixing, solar UVR-induced higher inhibition of photosynthetic rate in IS than in WS cells. During long-term (10 days) exposures to solar radiation, the specific growth rate was much lower in IS than WS at the beginning, then increased 3 days later to reach an equivalent level as that of WS. UVR-induced inhibition of photosynthetic carbon fixation in the IS was identical with that of WS at the end of the long-term exposure. The photosynthetic acclimation was not accompanied with increased contents of UV-absorbing compounds, indicating that repair processes for UVR-induced damages must have been accelerated or upgraded. (C) 2008 Elsevier B.V. All rights reserved.

Characterization of diurnal photosynthetic rhythms in the marine diatom Skeletonema costatum grown in synchronous culture under ambient and elevated CO2

Photosynthetic performance was examined in Skeletonema costatum (Greville) Cleve. under 12: 12-h light: dark (LD) cycle at ambient CO2 (350 muL L-1) and elevated CO2 (1000 muL L-1). At ambient CO2, the cellular chlorophyll a content, the light-saturated photosynthetic rate (P-m), the initial slope of the light saturation curves ( a), the photochemical efficiency of PSII (F-v/F-m), the apparent carboxylating efficiency (ACE) and the photosynthetic affinity for CO2 [1/K-m (CO2)] all showed rhythmical changes with different amplitudes during the light period. The P-m had similar changing pattern in the light period with the ACE and 1/K-m (CO2) rather than with the alpha and F-v/F-m, indicating that rhythmical changes of photosynthetic capacity may be mainly controlled by the activity of C- reduction associated with CO2 uptake during the light period. The CO2 enrichment reduced the ACE and the affinity to CO2, and increased the a, cellular chlorophyll a content and P m based on cell number. By contrast, the changing patterns of all photosynthetic parameters examined here during the light period had almost the same for cells grown at ambient CO2 and elevated CO2, suggesting that the photosynthetic rhythms of S. costatum are not affected by CO2 enrichment.

Effect of CO2 concentrations on the activity of photosynthetic CO2 fixation and extracelluar carbonic anhydrase in the marine diatom Skeletonema costatum

The growth and activity of photosynthetic CO2 uptake and extracellular carbonic anhydrase (CA(ext)) of the marine diatom Skeletonema costatum were investigated while cultured at different levels of CO2 in order to see its physiological response to different CO2 concentrations under either a low (30 mumol . m(-2) . s(-1)) or high (210 mumol . m(-2) . s(-1)) irradiance. The changes in CO2 concentrations (4-31 mumol/L) affected the growth and net photosynthesis to a greater extent under the low than under the high light regime. CAext was detected in the cells grown at 4 mumol/L CO2 but not at 31 and 12 mumol/L CO2, with its activity being about 2.5-fold higher at the high than at the low irradiance. Photosynthetic CO2 affinity (1/K-1/2(CO2)) of the cells decreased with increased CO2 concentrations in culture. The cells cultured under the high-light show significantly higher photosynthetic CO2 affinity than those grown at the low-light level. It is concluded that the regulations of CA(ext) activity and photosynthetic CO2 affinity are dependent not only on CO2 concentration but also on light availability, and that the development of higher CA(ext) activity and CO2 affinity under higher light level could sufficiently support the photosynthetic demand for CO2 even at low level of CO2.

Three species distribution models for the marine diatom Fragilariopsis kerguelensis of the Southern Ocean, links to NetCDF files

Fragilariopsis kerguelensis, a dominant diatom species throughout the Antarctic Circumpolar Current, is coined to be one of the main drivers of the biological silicate pump. Here, we study the distribution of this important species and expected consequences of climate change upon it, using correlative species distribution modeling and publicly available presence-only data. As experience with SDM is scarce for marine phytoplankton, this also serves as a pilot study for this organism group. We used the maximum entropy method to calculate distribution models for the diatom F. kerguelensis based on yearly and monthly environmental data (sea surface temperature, salinity, nitrate and silicate concentrations). Observation data were harvested from GBIF and the Global Diatom Database, and for further analyses also from the Hustedt Diatom Collection (BRM). The models were projected on current yearly and seasonal environmental data to study current distribution and its seasonality. Furthermore, we projected the seasonal model on future environmental data obtained from climate models for the year 2100. Projected on current yearly averaged environmental data, all models showed similar distribution patterns for F. kerguelensis. The monthly model showed seasonality, for example, a shift of the southern distribution boundary toward the north in the winter. Projections on future scenarios resulted in a moderately to negligibly shrinking distribution area and a change in seasonality. We found a substantial bias in the publicly available observation datasets, which could be reduced by additional observation records we obtained from the Hustedt Diatom Collection. Present-day distribution patterns inferred from the models coincided well with background knowledge and previous reports about F. kerguelensis distribution, showing that maximum entropy-based distribution models are suitable to map distribution patterns for oceanic planktonic organisms. Our scenario projections indicate moderate effects of climate change upon the biogeography of F. kerguelensis.

Effects of pCO2 and iron on the elemental composition and cell geometry of the marine diatom Pseudo-nitzschia pseudodelicatissima//(Bacillariophyceae)

Partial pressure of CO2 (pCO2) and iron availability in seawater show corresponding changes due to biological and anthropogenic activities. The simultaneous change in these factors precludes an understanding of their independent effects on the ecophysiology of phytoplankton. In addition, there is a lack of data regarding the interactive effects of these factors on phytoplankton cellular stoichiometry, which is a key driving factor for the biogeochemical cycling of oceanic nutrients. Here, we investigated the effects of pCO2 and iron availability on the elemental composition (C, N, P, and Si) of the diatom Pseudo-nitzschia pseudodelicatissima (Hasle) Hasle by dilute batch cultures under 4 pCO2 (~200, ~380, ~600, and ~800 µatm) and five dissolved inorganic iron (Fe'; ~5, ~10, ~20, ~50, and ~100 pmol /L) conditions. Our experimental procedure successfully overcame the problems associated with simultaneous changes in pCO2 and Fe' by independently manipulating carbonate chemistry and iron speciation, which allowed us to evaluate the individual effects of pCO2 and iron availability. We found that the C:N ratio decreased significantly only with an increase in Fe', whereas the C:P ratio increased significantly only with an increase in pCO2. Both Si:C and Si:N ratios decreased with increasing pCO2 and Fe'. Our results indicate that changes in pCO2 and iron availability could influence the biogeochemical cycling of nutrients in future oceans with high- CO2 levels, and, similarly, during the time course of phytoplankton blooms. Moreover, pCO2 and iron availability may also have affected oceanic nutrient biogeochemistry in the past, as these conditions have changed markedly over the Earth's history.

Seawater carbonate chemistry and silica during an experiment with a marine diatom Thalassiosira weissflogii, 2004

Opal accumulation rates in sediments have been used as a proxy for carbon flux, but there is poor understanding of the factors that regulate the Si quota of diatoms. Natural variation in silicon isotopes (delta.lc.gif - 54 Bytes30Si) in diatom frustules recovered from sediment cores are an alternative to opal mass for reconstructing diatom Si use and potential C export over geological timescales. Understanding the physiological factors that may influence the Si quota and the delta.lc.gif - 54 Bytes30Si isotopic signal is vital for interpreting biogenic silica as a paleoproxy. We investigated the influence of pCO2 on the Si quota, fluxes across the cell membrane, and frustule dissolution in the marine diatom Thalassiosira weissflogii and determined the effect that pCO2 has on the isotopic fractionation of Si. We found that our Si flux estimates mass balance and, for the first time, describe the Si budget of a diatom. The Si quota rose in cells grown with low pCO2 (100 ppm) compared with controls (370 ppm), and the increased quota was the result of greater retention of Si (i.e., lower losses of Si through efflux and dissolution). The ratio of efflux : influx decreased twofold as pCO2 decreased from 750 to 100 ppm. The efflux of silicon is shown to significantly bias measurements of silica dissolution rates determined by isotope dilution, but no effect on the Si isotopic enrichment factor (epsilon.lc.gif - 51 Bytes) was observed. The latter effect suggests that silicon isotopic discrimination in diatoms is set by the Si transport step rather than by the polymerization step. This observation supports the use of the v signal of biogenic silica as an indicator of the percentage utilization of silicic acid.

Seawater carbonate chemistry, growth rate and light sensitivity of marine diatom Chaetoceros muelleri (strain CSIRO CS-176) during experiments, 2011

This study investigated the impact of photon flux and elevated CO2 concentrations on growth and photosynthetic electron transport on the marine diatom Chaetoceros muelleri and looked for evidence for the presence of a CO2-concentrating mechanism (CCM). pH drift experiments clearly showed that C. muelleri has the capacity to use bicarbonate to acquire inorganic carbon through one or multiple CCMs. The final pH achieved in unbuffered cultures was not changed by light intensity, even under very low photon flux, implying a low energy demand of bicarbonate use via a CCM. In short-term pH drift experiments, only treatment with the carbonic anhydrase inhibitor ethoxyzolamide (EZ) slowed down the rise in pH considerably. EZ was also the only inhibitor that altered the final pH attained, although marginally. In growth experiments, CO2 availability was manipulated by changing the pH in closed flasks at a fixed dissolved inorganic carbon (DIC) concentration. Low-light-treated samples showed lower growth rates in elevated CO2conditions. No CO2 effect was recorded under high light exposure. The maximal photosynthetic capacity, however, increased with CO2 concentration in saturating, but not in subsaturating, light intensities. Growth and photosynthetic capacity therefore responded in opposite ways to increasing CO2 availability. The capacity to photoacclimate to high and low photon flux appeared not to be affected by CO2treatments. However, photoacclimation was restricted to growth photon fluxes between 30 and 300 µmol photons m-2 s-1. The light saturation points for photosynthetic electron transport and for growth coincided at 100 µmol photons m-2 s-1. Below 100 µmol photons m-2 s-1 the light saturation point for photosynthesis was higher than the growth photon flux (i.e. photosynthesis was not light saturated under growth conditions), whereas at higher growth photon flux, photosynthesis was saturated below growth light levels.

Physiological responses of the marine diatom Thalassiosira pseudonana to increased pCO2 and seawater acidity

We studied the effects of elevated CO2 concentration and seawater acidity on inorganic carbon acquisition, photoinhibition and photoprotection as well as growth and respiration in the marine diatom Thalassiosira pseudonana. After having grown under the elevated CO2 level (1000 µatm, pH 7.83) at sub-saturating photosynthetically active radiation (PAR, 75 µmol photons/m**2/s) for 20 generations, photosynthesis and dark respiration of the alga increased by 25% (14.69 ± 2.55 fmol C/cell/h) and by 35% (4.42 ± 0.98 fmol O2/cell/h), respectively, compared to that grown under the ambient CO2 level (390 µatm, pH 8.16), leading to insignificant effects on growth (1.09 ± 0.08 (1/d))v 1.04 ± 0.07 (1/d)). The photosynthetic affinity for CO2 was lowered in the high-CO2 grown cells, reflecting a down-regulation of the CO2 concentrating mechanism (CCM). When exposed to an excessively high level of PAR, photochemical and non-photochemical quenching responded similarly in the low- and high-CO2 grown cells, reflecting that photoinhibition was not influenced by the enriched level of CO2. In T. pseudonana, it appeared that the energy saved due to the down-regulated CCM did not contribute to any additional light stress as previously found in another diatom Phaeodactylum tricornutum, indicating differential physiological responses to ocean acidification between these two diatom species.

Short- and long-term conditioning of a temperate marine diatom community to acidification and warming

Ocean acidification and greenhouse warming will interactively influence competitive success of key phytoplankton groups such as diatoms, but how long-term responses to global change will affect community structure is unknown. We incubated a mixed natural diatom community from coastal New Zealand waters in a short-term (two-week) incubation experiment using a factorial matrix of warming and/or elevated pCO2 and measured effects on community structure. We then isolated the dominant diatoms in clonal cultures and conditioned them for 1 year under the same temperature and pCO2 conditions from which they were isolated, in order to allow for extended selection or acclimation by these abiotic environmental change factors in the absence of interspecific interactions. These conditioned isolates were then recombined into 'artificial' communities modelled after the original natural assemblage and allowed to compete under conditions identical to those in the short-term natural community experiment. In general, the resulting structure of both the unconditioned natural community and conditioned 'artificial' community experiments was similar, despite differences such as the loss of two species in the latter. pCO2 and temperature had both individual and interactive effects on community structure, but temperature was more influential, as warming significantly reduced species richness. In this case, our short-term manipulative experiment with a mixed natural assemblage spanning weeks served as a reasonable proxy to predict the effects of global change forcing on diatom community structure after the component species were conditioned in isolation over an extended timescale. Future studies will be required to assess whether or not this is also the case for other types of algal communities from other marine regimes.

Bacterial community structure of the marine diatom Haslea ostrearia

The marine diatom Haslea ostrearia produces a water-soluble blue-pigment named marennine of economic interest (e.g. in aquaculture for the greening of oysters). Up to date the studies devoted to ecological conditions under which this microalga develops never took into account the bacterial-H. ostrearia relationships. In this study the bacterial community was analysed by PCR-TTGE before and after H. ostrearia isolation cells recovered from 4 localities, to distinguish the relative part of the biotope and the biocenose and eventually to describe the temporal dynamic of the structure of the bacterial community. The bacterial structure of the phycosphere differed strongly from that of the bulk sediment. The similarity between bacteria recovered from the biofilm and the suspended bacteria did not exceed 10% (vs. > 90% amongst biofilms). The differences in genetic fingerprints, more especially high between two H. ostrearia isolates showed also the highest differences in the bacterial structure as the result of specific metabolomics profiles. The non-targeted metabolomic investigation showed that these profiles were more distinct in case of bacteria-alga associations than for the H. ostrearia monoculture. At the scale of a culture cycle in laboratory conditions, the bacterial community was specific to the growth stage. When H. ostrearia was subcultured for 9 months, a shift in the bacterial structure was shown from 3-months subculturing and the bacterial structure stabilized afterwards (70-86% similarities). A first insight of the relationships between H. ostrearia and its surrounding bacteria was shown for a better understanding of the ecological feature of this diatom.