Aubrey K. Davis

The place of diatoms in the biofuels industry

In spite of attractive attributes, diatoms are underrepresented in research and literature related to the development of microalgal biofuels. Diatoms are highly diverse and have substantial evolutionarily-based differences in cellular organization and metabolic processes relative to chlorophytes. Diatoms have tremendous ecological success, with typically higher productivity than other algal classes, which may relate to cellular factors discussed in this review. Diatoms can accumulate lipid equivalently or to a greater extent than other algal classes, and can rapidly induce triacylglycerol under Si limitation, avoiding the detrimental effects on photosynthesis, gene expression and protein content associated with N limitation. Diatoms have been grown on production scales for aquaculture for decades, produce value-added products and are amenable to omic and genetic manipulation approaches. In this article, we highlight beneficial attributes and address potential concerns of diatoms as biofuels research and production organisms, and encourage a greater emphasis on their development in the biofuels arena.

SYNCHRONIZED GROWTH OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) PROVIDES NOVEL INSIGHTS INTO CELL-WALL SYNTHESIS PROCESSES IN RELATION TO THE CELL CYCLE1

Cell‐cycle effects in phytoplankton have both general and specific influences over a variety of cellular processes. Understanding these effects requires that the majority of cells in a culture are progressing through the same cell‐cycle stage, which requires synchronous growth. We report the development of a silicon starvation–recovery synchrony for the first diatom with a sequenced genome, Thalassiosira pseudonana Hasle et Heimdale, which provides several novel insights into the process of cell‐wall formation. After 24 h of silicate starvation, flow cytometry measurements indicated that 80% of the cells were arrested in the early G1 phase of the cell cycle and then upon silicate replenishment progressed synchronously through the cycle. An early G1‐arrest point was not previously documented in diatoms. After silicate replenishment, girdle‐band synthesis was confined to a particular period in G1, and cells did not lengthen in accordance with each girdle band added, which has implications related to cell growth and separation processes in diatoms. Measurements of silicic acid uptake, intracellular pools, and silica incorporation into the cell wall, coupled with fluorescence visualization of newly synthesized cell‐wall structures, provide the first direct measurements of silica amounts in individual girdle bands and valves in a diatom. Fluorescence imaging indicated why valves in T. pseudonana do not have to reduce in size with each generation and enabled visualization of intermediates in structure formation. The development of a synchrony procedure for T. pseudonana enables correlation of cellular events with the cell cycle, which should facilitate the use of genomic information.

Gene Expression Induced by Copper Stress in the Diatom Thalassiosira pseudonana

ABSTRACT Utilizing a PCR-based subtractive cDNA approach, we demonstrated that the marine diatom Thalassiosira pseudonana exhibits a rapid response at the gene level to elevated concentrations of copper and that this response attenuates over 24 h of continuous exposure. A total of 16 copper-induced genes were identified, 11 of which were completely novel; however, many of the predicted amino acid sequences had characteristics suggestive of roles in ameliorating copper toxicity. Most of the novel genes were not equivalently induced by H2O2- or Cd-induced stress, indicating specificity in response. Two genes that could be assigned functions based on homology were also induced under conditions of general cellular stress. Half of the identified genes were located within two inverted repeats in the genome, and novel genes in one inverted repeat had mRNA levels induced by ∼500- to 2,000-fold by exposure to copper for 1 h. Additionally, some of the inverted repeat genes demonstrated a dose-dependent response to Cu, but not Cd, and appear to belong to a multigene family. This multigene family may be the diatom functional homolog of metallothioneins.

Metabolic and cellular organization in evolutionarily diverse microalgae as related to biofuels production

Microalgae are among the most diverse organisms on the planet, and as a result of symbioses and evolutionary selection, the configuration of core metabolic networks is highly varied across distinct algal classes. The differences in photosynthesis, carbon fixation and processing, carbon storage, and the compartmentation of cellular and metabolic processes are substantial and likely to transcend into the efficiency of various steps involved in biofuel molecule production. By highlighting these differences, we hope to provide a framework for comparative analyses to determine the efficiency of the different arrangements or processes. This sets the stage for optimization on the based on information derived from evolutionary selection to diverse algal classes and to synthetic systems.

A STRESS‐INDUCED PROTEIN ASSOCIATED WITH THE GIRDLE BAND REGION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYTA)1

We report the characterization of a cell‐surface protein isolated from the centric diatom Thalassiosira pseudonana Hasle and Heimdal. This protein has an apparent molecular weight of 150 kDa, is highly acidic, and is intimately associated with the cell wall. Although originally identified in cells experiencing copper toxicity, it is also induced by silicon and iron limitation but not by phosphate or nitrate limitation. Using immunofluorescence techniques, the 150‐kDa protein was localized to the girdle band region and covered the elongated girdle band region of morphologically aberrant cells suffering from copper toxicity. Although having biochemical similarities to girdle band associated proteins identified in pennate diatoms known as pleuralins, the 150‐kDa protein is not a sequence homolog and is predicted to have a number of unique features, such as chitin binding domains and a possible RGD cell attachment motif. Results presented here suggest that this protein is normally cell cycle regulated and may be involved in stabilizing cells during the division process.

Transcript level coordination of carbon pathways during silicon starvation‐induced lipid accumulation in the diatom Thalassiosira pseudonana

Summary Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity. To characterize the transcript level component of metabolic regulation, genome‐wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time‐course of silicon starvation. Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, and photosynthetic parameters were characterized alongside lipid accumulation. Extensive coordination of large suites of genes was observed, highlighting the existence of clusters of coregulated genes as a key feature of global gene regulation in T. pseudonana. The identity of key enzymes for carbon metabolic pathway inputs (photosynthesis) and outputs (growth and storage) reveals these clusters are organized to synchronize these processes. Coordinated transcript level responses to silicon starvation are probably driven by signals linked to cell cycle progression and shifts in photophysiology. A mechanistic understanding of how this is accomplished will aid efforts to engineer metabolism for development of algal‐derived biofuels.

Applications of Imaging Flow Cytometry for Microalgae

The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the diversity of cell types in natural samples, or diverse cellular responses in clonal populations, especially using imaging cytometry. Using examples from our laboratory, we review applications of imaging cytometry, specifically using an Amnis(®) ImageStream(®)X instrument, to characterize photosynthetic microalgae. Some of these examples highlight advantages of imaging flow cytometry for certain research objectives, but we also include examples that would not necessarily require imaging and could be performed on a conventional cytometer to demonstrate other concepts in cytometric evaluation of microalgae. We demonstrate the value of these approaches for (1) analysis of populations, (2) documentation of cellular features, and (3) analysis of gene expression.

Morphological Factors Involved in Adhesion of Acid-Cleaned Diatom Silica

PurposeDiatoms, unicellular microalgae with silica cell walls, have strong adhesive properties, which are dominated by chemical interactions between secreted organic material and the substrate. Possible technological applications of diatoms are likely to involve the adhesion of silica particles, or derivatives, which have been cleaned of organic material. Because the morphologies of diatom cell walls are far more complex than defined model structures, the relationship between morphology and adhesion for such materials is unknown.MethodsIn this paper we develop a new approach to monitor the adhesion of acid-cleaned diatom silica using parallel-plate flow chambers. We have evaluated factors such as settling time, extent of dryness, and substrate properties, and compared diatom species with silica features differing in size, shape, and percentage of surface contact area.ResultsResults indicated better adhesion of particles with higher surface contact area below a threshold of overall size, and a contribution by the number of possible contact surfaces to initial adhesion. We identified two stages in adhesion response to increasing shear stress. In the first stage, at low shear stress, species-dependent morphology played a major role in determining the strength of adhesion. After loosely adhered particles were removed at low shear, a second stage of persistent adhesion emerged at higher shear stresses. In the second stage, variations in morphology had a much smaller effect on adhesion.ConclusionsThese results identify conditions and fundamental morphological features for adhesion that can be utilized in future technological applications of silica particles with complex shapes.

Diatoms and diatomaceous earth as novel poultry vaccine adjuvants.

&NA; Diatoms are single cell eukaryotic microalgae; their surface possesses a porous nanostructured silica cell wall or frustule. Diatomaceous earth (DE) or diatomite is a natural siliceous sediment of diatoms. Since silica has been proved to have adjuvant capabilities, we propose that diatoms and DE may provide an inexpensive and abundant source of adjuvant readily available to use in livestock vaccines. In a first experiment, the safety of diatoms used as an adjuvant for in‐ovo vaccination was investigated. In a second experiment, we assessed the humoral immune response after one in‐ovo vaccination with inactivated Newcastle Disease Virus (NDV) and DE as adjuvant followed by 2 subcutaneous boosters on d 21 and 29 of age. In both experiments, results were compared to Freunds incomplete adjuvant and aluminum hydroxide. No detrimental effects on hatchability and chick quality were detected after in‐ovo inoculation of diatoms and DE in experiments 1 and 2 respectively. In experiment 2 no humoral responses were detected after the in‐ovo vaccination until 29 d of age. Seven d after the second subcutaneous booster an antibody response against NDV was detected in chickens that had received vaccines adjuvanted with Freunds incomplete adjuvant, aluminum hydroxide, and DE. These responses became significantly higher 10 d after the second booster. Finally, 15 d after the second booster, the humoral responses induced by the vaccine with Freunds incomplete adjuvant were statistically higher, followed by comparable responses induced by vaccines containing DE or aluminum hydroxide that were significantly higher than DE+PBS, PBS+INDV and PBS alone. From an applied perspective, we can propose that DE can serve as a potential adjuvant for vaccines against poultry diseases.

Clarification of Photorespiratory Processes and the Role of Malic Enzyme in Diatoms

Evidence suggests that diatom photorespiratory metabolism is distinct from other photosynthetic eukaryotes in that there may be at least two routes for the metabolism of the photorespiratory metabolite glycolate. One occurs primarily in the mitochondria and is similar to the C2 photorespiratory pathway, and the other processes glycolate through the peroxisomal glyoxylate cycle. Genomic analysis has identified the presence of key genes required for glycolate oxidation, the glyoxylate cycle, and malate metabolism, however, predictions of intracellular localization can be ambiguous and require verification. This knowledge gap leads to uncertainties surrounding how these individual pathways operate, either together or independently, to process photorespiratory intermediates under different environmental conditions. Here, we combine in silico sequence analysis, in vivo protein localization techniques and gene expression patterns to investigate key enzymes potentially involved in photorespiratory metabolism in the model diatom Thalassiosira pseudonana. We demonstrate the peroxisomal localization of isocitrate lyase and the mitochondrial localization of malic enzyme and a glycolate oxidase. Based on these analyses, we propose an updated model for photorespiratory metabolism in T. pseudonana, as well as a mechanism by which C2 photorespiratory metabolism and its associated pathways may operate during silicon starvation and growth arrest.