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Recent development of DNA sequencing technologies has enabled successful construction of thousands of mitochondrial genomes. Nevertheless, only 33 mitochondrial genomes have been reported for species in Bacillariophyta, which includes harmful algal bloom (HAB) species. In this study, we determined the complete mitogenome of the Bacillariophyta HAB species Odontella regia. For comparison, we also constructed the mitogenome of Lithodesmium undulatum, another Bacillariophyta species. Both strains were isolated in the Jiaozhou Bay, an epitome of China’s coastal ecosystem and an ideal site for HAB research. These two mitogenomes were characterized as 37,617 bp and 37,057 bp circular-mapping molecules with AT content of 73.4% and 75.3%, respectively. The phylogenetic tree based on 32 protein-coding genes of the mitogenome encoded in 35 Bacillariophyta species revealed that the class Mediophyceae consisted of multi-phyletic clades. While O. regia formed an independent clade, L. undulatum was closely related to Thalassiosira pseudonana and Skeletonema marinoi. Synteny comparison of O. regia and L. undulatum mitogenomes and mitogenomes of three closely related species displayed substantial differences among lineages in Mediophyceae by a series of gene translocations and/or inversion with many conserved gene blocks such as rpl2-rps19-rps3-rpl16-atp9. These analyses suggested a complex evolutionary relationship in Mediophyceae in which the mitogenome of O. regia was the least conservative compared with the mitogenomes of four other Bacillariophyta species. Further studies are needed to clarify detailed phylogenetic relationships in Bacillariophyta.

Global warming is altering both phytoplankton-experienced temperature and light-exposure duration through shifting their niches from low to high latitudes. We explored the growth, physiology, and compositions of a smaller Thalassiosira pseudonana and a larger Thalassiosira punctigera, temperate marine centric diatoms, in responses to a matrix of temperatures (12, 15, 18, and 21 °C) and photoperiods (light:dark cycles of 4:20, 8:16, 16:8, and 24:0). Both T. pseudonana and T. punctigera grew faster under medium temperature and longer photoperiod, under the expected optimal instantaneous light intensity. The biovolume-based pigments content of T. pseudonana responded largely to temperature, while that of T. punctigera responded more to photoperiod duration than to temperature. In T. pseudonana, shortest photoperiod enhanced cellular protein content and alleviated their temperature dependency. Continuous growth light reduced the photosynthetic capacity of T. pseudonana at the lowest temperature and reduced that of T. punctigera across temperatures. Moreover, we found the increasing temperature linearly increased the dark respiration rate (Rd) and molar ratio of carbon to nitrogen (C:N) of T. pseudonana but decreased that of T. punctigera, with the scattered effects of photoperiod. Our results demonstrated that responses of diatoms Thalassiosira across photoperiods and temperatures vary with species and possibly with cell size, suggesting that the poleward shift of the niches of phytoplankton in nature might cause a change in community structure.

The diurnal solar cycle is the single most common influential variable for algae growing in a natural environment. In spite of its importance, relatively few laboratory studies have been directed towards photophysiological responses to dynamic irradiance regimes. Further, most of these studies have utilized optically thin cultures. However, phycological technologies utilize optically dense cultures grown in depths measured by centimeters. In this case, algae are harvested under self-shading conditions (all usable incident irradiance being absorbed), and the response of the entire water column to the dynamic irradiance regime is of interest. Thus, the area integrated under some modeled curve for a dynamic irradiance regime becomes the independent variable for the photobiological response being measured of a water column in laboratory studies, and this is important. But what curve should be used to model a dynamic irradiance regime? We suggest that a Gaussian (normal) distribution be used to model this dynamic irradiance because of its well-known statistical attributes. In this study, Thalassiosira pseudonana (marine diatom) was grown under shade-limited growth conditions in a 20-cm deep water column with a dynamic irradiance regime using our SolarStat™. A Gaussian distribution modeled this regime having an 11-h day length (standard deviation ±1.85 h) with an irradiance maximum of 1900 μmol photons m−2 s−1. The daily productivity of batch culture in the linear phase of the light-controlled growth dynamic is compared with those of semi-continuous cultures maintained at 1.0, 0.5, and 0.25 doublings day−1.

There is relatively little choice in cultivation methods for growing algae outdoors, either in open pond systems or closed photobioreactors—as batch, continuous, or semi-continuous culture. Algal batch culture grown in a nutrient replete environment with adequate sunlight will become self-shaded with sufficient cell density and enter a stage in the growth dynamic known as the “phase of linear growth.” It is during this phase of linear growth that primary production is at maximum and that the highest biomass is harvested. The inherent problem with batch culture is that the exponential (and possibly lag) phases necessary to achieve densities required prior to the phase of linear growth consume time and waste surface area, and thereby make this an inefficient method to grow algae. Semi-continuous culture can be forced into shade-limiting conditions by reducing growth rate from maximum through dilution, whereby phases of lag and exponential growth are skipped, and culture growth is put into a state similar to a perpetual phase of linear growth with an appropriate culture harvest/dilution cycle. Importantly, semi-continuous culture can increase net growth efficiency over batch culture when compared by shade-limited growth rate. However, scientific study and theory covering shade-limited algal growth under semi-continuous culture conditions are nearly non-existent, which currently makes its application to phycological technologies impractical through “hit and miss” strategies. This laboratory study compares shade-limited growth dynamics for batch and semi-continuous cultures of Thalassiosira pseudonana (small-sized, marine diatom). Theory for optimizing production of mass algal culture with semi-continuous culture technique through cycle period and harvest volume is developed, and guidelines to practical industrial applications are provided.

The health beneficial omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are naturally synthesized by diatoms through consecutive steps of fatty acid elongase and desaturase enzymes. In Thalassiosira pseudonana, these fatty acids constitute about 10–20 % of the total fatty acids, with EPA accumulation being five to ten times higher than DHA. In order to identify the subcellular localization of enzymes in the pathway of LC-PUFA biosynthesis in T. pseudonana and to manipulate the production of EPA and DHA, we generated constructs for overexpressing each of the T. pseudonana long-chain fatty acid elongase genes. Full-length proteins were fused to GFP, and transgenic lines were generated. In addition, overexpressed native proteins with no GFP fusion were tested. The subcellular localization of each elongase protein was determined. We then examined the total amount of lipids and analyzed the fatty acid profile in each of the transgenic lines compared to wild type. Lines with overexpressed elongases showed an increase of up to 1.4-fold in EPA and up to 4.5-fold in DHA, and the type of fatty acid that was increased (EPA vs. DHA) depended on the type of elongase that was overexpressed. This data informs future metabolic engineering approaches to further improve EPA and DHA content in diatoms.

We hypothesize that, in algae, carbon allocation to lipids depends on the combined effects of the metabolic constraints imposed by the genotype (i.e., species-specific differences in composition) and on the acclimation responses (phenotype) to changes in the stoichiometry of available inorganic carbon (for photosynthesis) and nitrogen (primarily for amino acids, protein, and nucleic acid synthesis). We thus cultured three taxonomically distinct algae, the green alga Dunaliella salina, the diatom Thalassiosira pseudonana, and the dinoflagellate Protoceratium reticulatum, at four nitrate concentrations and constant inorganic carbon. Since energy availability also directly impacts carbon partitioning, we studied the effect of irradiance on the oil quality of P. reticulatum. We used Fourier transform infrared (FTIR) spectroscopy to study carbon allocation and biomass reduction level and gas chromatography for fatty acid analysis. The fatty acid complements of the three species were different; within each species, growth conditions substantially altered oil quality. We ranked the oils in terms of their suitability as biodiesels, using international standards as reference. We believe that this approach may help to identify the appropriate combination of taxa and culture conditions for algal biodiesel production and in general offers insight on carbon allocation to fatty acids.

Investigation of the production and structural characteristics of the extracellular polysaccharides (ECPS) released by diatoms according to nutrient status is urgently necessary for our understanding of the roles of ECPS in aquatic ecosystem. In this study, the effects of N and P depletion and variable nutrient sources on the production and structural characterization of the soluble ECPS released by the marine diatom Thalassiosira pseudonana in batch culture were examined. The abundance of the soluble ECPS produced under N (NO3 −) depletion was higher than that obtained in the P (PO4 3−) depletion and control treatments. NH4 + rather than NO3 − and urea and sodium-glycerophosphate rather than PO4 3− were found to induce a higher abundance of soluble ECPS production at the end of the experiment, indicating that the abundance of soluble ECPS is also affected by the nutrient source. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of the structural characteristics of the soluble ECPS samples showed that the degree of polymerization (d.p.) profiles and the distribution of the polymers with different molecular masses varied in response to nutrient depletion and different nutrient sources. An increase in the proportion of both low- and high-molecular-weight polymers under the N (NO3 −) and P (PO4 3−) depletion treatments was found. Based on the different characteristics of high- and low-molecular-weight polymers, our results suggest that the production of soluble ECPS may be one of the strategies that T. pseudonana utilizes to survive under nutrient stress conditions.

Future algal biotechnology will need enhanced production strains, capable of more rapid growth, more efficient solar-energy conversion and/or higher levels of metabolite production. Almost certainly transgenic organisms will be used to ensure the cost-effective, economically viable production of a range of metabolites. As with all biotechnological processes, the functional stability, reliability and security of the production strains will be of paramount importance in algal biotechnology, as without this no biotechnological process is sustainable. In this study, the transgenic model strain Thalassiosira pseudonana CCAP 1085/23 was cryopreserved using a conventional, low-tech, colligative cryopreservation protocol. This employed dimethyl sulphoxide [5 % (v/v)] as a cryoprotectant, using a two-step cooling approach: initial controlled-rate cooling, followed by plunging into liquid nitrogen. High levels of post-thaw viability (70–85 %) were obtained, and on recovery of cryopreserved material no reduction in expression of the protein of the inserted gene (big1-GFP) was observed. Additionally, cryopreservation does not affect the localisation of the BIG1-GFP protein as demonstrated by microscopy of stained samples, nor its functionality as demonstrated by Western blotting.

Although substantial economic barriers exist, marine diatoms such as Thalassiosira pseudonana and Phaeodactylum tricornutum hold promise as feedstock for biodiesel because of their ability to manufacture and store triacylglycerols (TAGs). The recent sequencing of these two marine diatom genomes by the United States Department of Energy Joint Genome Institute and the development of improved systems for genetic manipulation should allow a more systematic approach to understanding and maximizing TAG production. However, in order to best utilize these genomes and genetic tools, we must first gain a deeper understanding of the nutrient-mediated regulation of TAG anabolism. By determining both the yield and molecular species distribution of TAGs we will, in the future, be able to fully characterize the effects of genetic manipulation. Here, we lay the groundwork for understanding TAG production in T. pseudonana and P. tricornutum, as a function of nitrate and silicate depletion. Diatoms were starved of either nitrate or silicate, and TAGs were extracted with hexane from lyophilized samples taken at various time intervals following starvation. The timing of TAG production and the relative abundance of TAGs were estimated by fluorescence spectroscopy using Nile red and the total yield per biomass determined by gravimetric assay. TAGs were analyzed using thin layer chromatography, gas chromatography–mass spectrometry, and electrospray ionization mass spectrometry to identify the major TAG species produced during the growth curve. Under our conditions, the TAG yield from T. pseudonana is about 14–18% of total dry weight. The TAG yield from P. tricornutum is about 14% of total dry weight. Silicate-starved T. pseudonana accumulated an average of 24% more TAGs than those starved for nitrate; however, the chemotypes of the TAGs produced were generally similar regardless of the starvation condition employed.

Nine microalgal species from the classes Bacillariophyceae, Cryptophyceae, Prymnesiophyceae and Dinophyceae were isolated from Australian waters, cultured to stationary phase and analyzed for their lipid and fatty acid composition and yield. Five species (Pavlova pinguis, Heterocapsa niei, Proteomonas sulcata, Navicula jeffreyi and Thalassiosira pseudonana) produced high proportions of triacylglycerol (TAG: 22–57% total lipid). An unidentified Navicula-like diatom (CS-786), despite having a low TAG content, had the highest EPA yield (5.8 mg L−1), due to high biomass and a high relative proportion of EPA. Heterocapsa niei had the highest DHA yield (2.9 mg L−1), due to a high cellular lipid and DHA content (171 pg cell−1 and 13.7 pg cell−1, respectively) despite its relatively low biomass. The desirable PUFA composition and yield of both diatom CS-786 and H. niei make them potential candidates for optimization of biomass and PUFA production for use as live-feeds in aquaculture. In addition, H. niei may have potential as a source of DHA for other uses. Low proportions (< 1.2%) of 24:6(n−3) accompanied by trace proportions of 24:5(n−6) were detected in most strains, while 28:8(n−3) was found in dinoflagellates and also in the prymnesiophyte P. pinguis. All non-diatomaceous species contained 26:7(n−3) in minor quantities. This is the first time these unusual C24 and C26 PUFA have been reported in microalgae and the first report of C28 PUFA in a microalga other than dinoflagellates. Possible biosynthetic reasons why these might occur in stationary phase cultures are considered and the likely dietary transfer of these PUFA to higher aquatic life is discussed.

Chemical composition of the microalga Thalassiosira pseudonana Hasle & Heimdalwas studied with different proton nuclearmagnetic resonance (1H NMR)techniques, and by comparing NMR spectrafrom extraction samples with a spectrumfrom a sample of whole cells we show thathigh-resolution magic angle spinning (HRMAS) 1H NMR can be used for broadrange analysis of metabolic composition inmicroalgal whole cells. Signals fromimportant metabolites such aspolyunsaturated fatty acids (PUFAs)eicosapentaenoic (EPA) and docosahexaenoic(DHA) acids were seen in a 1H NMRspectrum of lipophilic extract, andpossibly also signals from the carotenoidfucoxanthin. In a spectrum of hydrophilicextract we assigned signals to amino acidssuch as glutamine (Gln) and glutamic acid(Glu), carbohydrate and ATP. These findingswere compared to a spectrum of HR MAS1H NMR analysis of whole cells, whereit was possible to find signals coincidentwith the different metabolites seen inspectra of the extraction samples. Sincethe position of resonance peaks in a NMRspectrum depends on the chemicalsurroundings of each atom at the time ofanalysis some peak shift differencesbetween extract and whole cell samplespectra may occur, but signal shifts werenot significantly different between theanalyses here. In addition, application ofHR MAS highly increased spectral resolutionin the complex whole cell sample. Wetherefore suggest that HR MAS 1H NMRanalysis is a suitable analysis tool tostudy metabolic composition directly onwhole cells of microalgae, making itpossible to study a broad range ofmetabolites simultaneously without tediousextraction procedures.

The vitamin content in four Australian microalgae, a Nannochloropsis-like sp., Pavlova pinguis, Stichococcus sp. and Tetraselmis sp., were examined. These were grown under a 12:12 h light:dark regimen (100 μmol photon m−2s−1) and harvested during late-logarithmic phase. Typically, the content showed a two- to three fold range between the species. When expressed on a dry weight basis, the content of ascorbate ranged from 1.3 to 3.0 mg g−1, β-carotene from 0.37 to 1.05 mg g−1, α-tocopherol from 0.07 to 0.29 mg g−1, thiamine from 29 to 109 μg g−1, riboflavin from 25 to 50 μg g−1, total folates from 17 to 24 μg g−1, pyridoxine from 3.6 to 17 μg g−1, cobalamin from 1.70 to 1.95 μg g−1 and biotin from 1.1 to 1.9 μg g−1. Retinol was detected only in Tetraselmis sp. (2.2 μg g−1); any vitamins D2 or D3 were below the detection limit (≤0.45 μg g−1). Nannochloropsis sp. was also grown under a 24:0 h light:dark light cycle and harvested at stationary phase. The content of most vitamins in Nannochloropsis sp. cultures differed significantly, and the degree of variation was similar to that observed between the four species grown under 12:12 h light:dark regimen (100 μmol photon m−2s−1) and harvested during late-logarithmic phase. Thiamine content was also examined in six non-Australian strains commonly used in aquaculture, Chaetoceros muelleri, Thalassiosira pseudonana, Nannochloris atomus, Nannochloropsis oculata, Isochrysis sp. (T.ISO) and Pavlova lutheri. Values (average 61 μg g−1; range 40 to 82) were similar to those in the Australian strains (average 61 μg g−1; range 29 to 109) and increased during stationary phase (average 94 μg g−1; 38 to 131). Comparison of the data with the known nutritional requirements for marine fish species and prawns suggests that the microalgae should provide excess or adequate levels of the vitamins for aquaculture food chains. The data may be used to guide the content of vitamins included in micro-diets developed as replacements for live diets.

The diatomsChaetoceros sp.,Skeletonema costatum andThalassiosira pseudonana were grown with different irradiances of white and of blue-green light, and with a mixture of blue-green plus 6.5 μmol m−2 s−1 of white light. Exponential growth rates were higher in mixed blue for the first two, whileT. pseudonana grew faster in white light but, in all cases, mean cell division rates did not differ with increasing irradiances. Harvesting in stationary, rather than in late exponential growth phase, resulted in higher protein contents forChaetoceros sp. andS. costatum, but forT. pseudonana the highest value was in the exponential phase. The highest protein content was in blue-green light for the three species and it increased with irradiance. As to other fractions, the three strains showed different responses, related to quality and quantity, as well as to culture ages.

Concentrations of riboflavin — a vitamin essential for maricultured animals—were measured in six species of microalgae commonly used in mariculture. These were two diatoms (Chaetoceros gracilis, Thalassiosira pseudonana); two prymnesiophytes (Isochrysis sp. (clone T.ISO),Pavlova lutheri); one chlorophyte (Nannochloris atomus) and one eustigmatophyte (Nannochloropsis oculata). Cultures were analysed during both logarithmic and stationary growth phase.

The ascorbic acid (vitamin C) concentrations in 11 species of microalgae commonly used in mariculture were determined. The species examined were 4 diatoms (Chaetoceros calcitrans (Paulsen) Takano,Chaetoceros gracilis Schutt,Skeletonema costatum (Greville) Cleve,Thalassiosira pseudonana (Hustedt, clone 3H) Hasle and Heimdal); 2 prymnesiophytes (Isochrysis sp. (clone T.ISO) Parke,Pavlova lutheri (Droop) Green); 1 prasinophyte (Tetraselmis suecica (Kylin) Butcher); 2 chlorophytes (Dunaliella tertiolecta Butcher,Nannochloris atomus Butcher); 1 eustigmatophyte (Nannochloropsis oculata (Droop) Green); and 1 cryptophyte (Chroomonas salina (Wislouch) Butcher). Duplicate cultures of each species were grown under defined conditions and analysed during both logarithmic and stationary phase of growth. Average values for ascorbic acid ranged from 9.4 fg cell−1 (N. oculata, stationary phase) to 700 fg cell−1 (S. costatum, stationary phase). This value was generally related to cell size. Levels of ascorbic acid cell−1 increased during the stationary growth phase forS. costatum andD. tertiolecta and decreased forC. gracilis, T. pseudonana, C. salina andN. oculata. Levels did not change significantly for the remaining species. Average values for per cent ascorbic acid ranged from 0.11% (T. pseudonana, stationary phase) to 1.62% of dry weight (C. gracilis, logarithmic phase). The per cent ascorbic acid was not related to algal class. Also, the percentage between logarithmic and stationary phase cultures differed for many of the species, but differences were unrelated to algal class.Chaetoceros gracilis, T. pseudonana, N. oculata andIsochrysis sp. (T.ISO) had higher per cent ascorbic acid during the logarithmic phase, whereasD. tertiolecta andN. atomus contained more per cent ascorbic acid during the stationary phase. Despite the differences in the composition of the different microalgae (0.11–1.62% ascorbic acid), all species would provide a rich source of ascorbic acid for maricultured animals, which can require 0.003–0.02% of the vitamin in their diet.