Algal Research

BackgroundPolyphosphates (polyP) and ATP are phosphate-containing metabolites present in prokaryotes and eukaryotes. PolyP has a wide variety of functions including phosphate and cation storage. ATP is a central metabolite in cellular bioenergetics and the phosphate providing substrate of polyP. In the green microalga Chlamydomonas reinhardtii, polyP synthesis is suggested to buffer ATP concentration, and the role of polyP in energetic metabolism requires further investigation. In this aim, relative quantification of both metabolites is needed. Because ATP and polyP half-lives differ greatly, harvesting generates biases in this relative quantification in current methods. For this reason, we present here a joint protocol optimised to compromise between maximal yield and specific constraints of both assays. MethodsThe optimised method quantifies ATP and polyP from the same C. reinhardtii cell extract after neutral phenol-chloroform extraction. Cells are directly pipetted from the culture to the phenol-chloroform- EDTA extraction mix. After a second chloroform extraction, ATP is quantified directly from the extract, while polyP measurement requires purification by ethanol precipitation. We used one-way analysis of variance or Kruskall-Wallis testing and appropriate post hoc testing to evaluate statistical effects in our results. ResultsWe show that he polyP/ATP ratio of the reference strain CC-4533 in exponential mixotrophic growth is around 65. While the optimised protocol performs as well as specific protocols for either ATP or polyP, the dispersion of the polyP/ATP ratio is twice better than for the separate metabolites. Direct sampling from the culture works better than centrifugation and filtration to maintain physiological conditions and high polyP yield. Using spiking with ATP and polyP, we show that ATP and longer chain polyP are fully recovered but not very short chain polyP. Finally, we show that the polyP chain length distribution extracted from CC-4533 is very broad, reaching up to several thousand P with a mean around 200 P. DiscussionOur protocol improves the precision of relative quantification of ATP and polyP by using neutral phenol-chloroform extraction and allows polyP/ATP ratio calculation from low-density samples and without normalisation. It can be applied to other microorganisms or cells, in a variety of physiological and stress conditions.

The polyextremophilic Cyanidiales are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal {beta}-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/530181v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@dd010dorg.highwire.dtl.DTLVardef@1700e5corg.highwire.dtl.DTLVardef@1bee8eaorg.highwire.dtl.DTLVardef@ad67da_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microalgae play a crucial role in ecosystems, from oxygen production to sustaining food webs and offering valuable applications in industry and environmental management. Increasing their productivity in terms of biomass yield, cultivation time, and nutritional or metabolite quality remains a major challenge. This study assessed the effects of 10 bioactive substances (including lactones, phytohormones, and natural extracts), four light wavelengths, and four CO2 injection regimes on Arthrospira platensis, Chlorella vulgaris, Ankistrodesmus falcatus, and Tetradesmus dimorphus, using linear modeling and LSD (Least Significant Difference) post hoc tests. N-butyryl-DL-homoserine lactone significantly enhanced A. platensis growth (94.4%), while naphthaleneacetic acid and indole-3-butyric acid promoted T. dimorphus growth (138.1% and 115.5%, respectively). More accessible alternatives, such as Aloe vera and coconut water, also stimulated growth: A. platensis, C. vulgaris, and A. falcatus increased by 85.3%, 69.2%, and 87.7% with Aloe vera, while T. dimorphus increased 80.5% with coconut water. Regarding light quality, red light (600-700 nm) benefited A. platensis and A. falcatus (49.2% and 20.8%, respectively), whereas blue light (400-490 nm) favored C. vulgaris and T. dimorphus (57.7% and 31.5%, respectively). CO2 injection further enhanced biomass production and carbon fixation, particularly in C. vulgaris and A. falcatus (73.5% and 53.5%, respectively). However, combined treatments did not produce additive effects, suggesting complex interactions. Overall, these findings demonstrate the potential of bioactive substances and environmental conditions to improve microalgal performance and highlight the importance of investigating synergistic effects and scalability for large-scale production.

Nitrogen is an important element for all living organisms. Photoautotrophic organisms need to assimilate nitrogen from the environment, therefore changes in nitrogen availability have a strong influence on their growth and metabolism. Many microalgae have been known to contain crystalline inclusions, and recently, it has been shown that many of these consist of purines like guanine and thus must be linked to the cellular nitrogen metabolisms. The alveolate alga Chromera velia contains such guanine crystals, and during its lifecycle, the alga is thought to be subjected to strong changes in external nitrogen availability. Here, we investigated the formation or decline of crystalline guanine in dependence of the availability of inorganic nitrogen in the growth medium. Cells were examined using polarised light microscopy, Raman micro-spectroscopy, chromatography (HPLC), transmission and scanning electron microscopy. The cellular guanine crystal content decreased during nitrogen starvation and increased upon transfer of the cells back to standard growth medium containing nitrate. Raman micro-spectroscopy showed that the crystals were composed of anhydrous guanine in beta-polytype. They appear in unspecific positions throughout the cell, and staining with the green dye Lysotracker DND-26 suggests that they are within vacuoles. Stacks of crystals could be observed in cells via freeze fracture and freeze etching electron microscopy, which unambiguously showed a membrane around the crystal aggregates, in a similar arrangement as has been shown for guanine storage vacuoles (GSV) in Chlamydomonas reinhardtii. We developed a method to isolate the guanine crystals from whole cells, and were able to obtain crystals which retained their flat, plate-like structure, matching the electron microscopic observations from whole cells. The isolated crystals were shown to consist of nitrogen rich compounds via energy-dispersive X-ray (EDX) analysis, and Raman micro-spectroscopy confirmed that they consist of guanine.

Mammalian cell culture technologies are crucial for recombinant protein production, organoid generation, medical applications, and the generation of in vitro cultivated meat. However, they are limited by high costs, vascular O2 provision, and the resultant inhibition of 3D tissue formation. Effective media usage along with oxygenation and waste management to extend culture health and longevity are key to improving all three. Microalgae, utilizing organic or inorganic CO2, produce O2 from light which complements oxygen-consuming and CO2-respiring mammalian cells and tissue culture. However, common microalgal cultivation conditions differ in temperature and salinity from mammalian cell cultivation environments, making co-cultivation short-lived and challenging. We screened several different microalgae species to identify locally isolated Chlorella BDH-1 as candidate that has high growth rates in mammalian culture conditions while, unlike other Chlorella species, does not compete for glucose as an energy source. In mammalian cell co-culture, BDH1 reduces cellular waste products, stabilizes pH, doubles culture longevity, increases growth performance up to 80%, and reduces expensive and ethically challenging foetal bovine serum requirements. Chlorella BDH-1 was also non-inflammatory and tolerant of clinical antibiotics. Collectively, mammalian cell/BDH1 co-cultivation improves tissue culture health and reduces costs, paving the path for applications in the biotechnology and medical sectors.

Achieving enhanced lipid yield without compromising biomass is one of the long-standing challenges in our quest to produce algal biofuel sustainably. Multiple factors, including temperature, nutrients and light conditions impact lipid production, however such lipid-enhancing strategies often lead to reduced biomass, thereby offsetting the total volume of lipid recovered. Hydrodynamic cues remain poorly studied, specifically in the context of lipid production in motile algae, concurrently with biomass generation and photo-physiology, a key fitness parameter. By imposing hydrodynamic cues to biophysically stress distinct strains of raphidophyte Heterosigma akashiwo at specific time points along the growth stages (indicating different nutritional states), we quantify the lipid production, alongside algal biomass and photo-physiology. Early induction (hydrodynamic cues implemented during the lag phase) and delayed induction (hydrodynamic cues implemented during the exponential phase) were studied. Delayed induction of hydrodynamic cues suppressed growth and photo-physiology without significant enhancement of lipid production, however, early induction allowed to significantly increase lipid content, up to 300%, without observable changes in biomass and photo-physiology. Based on this, we propose a hydrodynamic strategy for enhanced lipid production with sustained biomass and physiological fitness. This work presents hydrodynamic perturbation and its onset timing as tunable parameters to advance lipid production technologies across diverse motile species.

Microalgae are well-known for their role as sustainable bio-factories, offering a promising solution to the global food and nutrition crisis. To clarify the potential of Chlorella sorokiniana UTEX 1230 for food applications, particularly as an alternative protein source, the study employed a mixotrophic cultivation mode with sodium acetate (NaAc) as a cost-effective organic carbon (NaAc-C) source. Varying levels of NaAc-C and nitrate-sourced nitrogen were investigated, optimizing the effect of metabolic characteristics of the microalgal growth. The designed heterotrophic cultivation confirmed the ability of C. sorokiniana UTEX 1230 to grow on NaAc-C, and then the mixotrophic cultures, when supported by both NaAc-C and CO2, exhibited superior growth performance, achieving double the biomass concentration compared to the autotrophic control. The addition of nitrogen (750 mg/L NaNO) facilitated the thorough metabolism of NaAc-C and enhanced photosynthetic activity indicated by a 196% increase in pigment levels, which resulted in a maximum biomass concentration of 2.82 g/L in the 150 mM NaAc-C group. A detailed analysis of nitrogen and protein concentrations over time revealed that higher nitrogen availability led to greater protein accumulation which was then degraded to support essential life activities under nitrogen starvation. Therefore, it is suggested that supplementing nitrate on the 3rd day and harvesting on the 4th day could be strategically implemented to increase protein yield from 0.17 g/L/d to 0.34 g/L/d. These findings offer theoretical guidance for further refining this microalgal strain for use as an alternative protein.

Determining seaweed protein concentration and the associated phenotype is critical for food industries that require precise tools to moderate concentration fluctuations and attenuate risks. Algal protein extraction and profiling have been widely investigated, but content determination involves a costly, time-consuming, and high-energy, laboratory-based fractionation technique. The present study examines the potential of field spectroscopy technology as a precise, high-throughput, non-destructive tool for on-site detection of red seaweed protein concentration. By using information from a large dataset of 144 Gracilaria sp. specimens, studied in a land-based cultivation set-up, under six treatment regimes during two cultivation seasons, and an artificial neural network, machine learning algorithm and diffuse visible-near infrared reflectance spectroscopy, predicted protein concentrations in the algae were obtained. The prediction results were highly accurate (R2 = 0.95; RMSE = 0.84), exhibiting a high correlation with the analytically determined values. External validation of the model derived from a separate trial, exhibited even better results (R2 = 0.99; RMSE = 0.45). This model, trained to convert phenotypic spectral measurements and pigment intensity into accurate protein content predictions, can be adapted to include diversified algae species and usages. HighlightNon-destructive determination of protein content in the edible red seaweed Gracilaria sp. by in-situ, VIS-NIR spectroscopy and a machine learning algorithm.

The west coast of Saudi Arabia borders the Red Sea, which maintains high average temperatures and increased salinity compared to other seas or oceans. Summer conditions in the Arabian Peninsula may exceed the temperature tolerance of most currently cultivated microalgae. The Cyanidiales are polyextremophilic red algae whose native habitats are at the edges of acidic hot springs. Cyanidioschyzon merolae 10D has recently emerged as an interesting model organism capable of high-cell density cultivation on pure CO2 with optimal growth at 42 {degrees}C and low pH between 0.5-2. C. merolae biomass has an interesting macromolecular composition, is protein rich, and contains valuable bio-products like heat-stable phycocyanin, carotenoids, {beta}-glucan, and starch. Here, photobioreactors were used to model C. merolae 10D growth performance in simulated environmental conditions of the mid-Red Sea coast across four seasons, it was then grown at various scales outdoors in Thuwal, Saudi Arabia during the Summer of 2022. We show that C. merolae 10D is amenable to cultivation with industrial-grade nutrient and CO2 inputs outdoors in this location and that its biomass is relatively constant in biochemical composition across culture conditions. We also show the adaptation of C. merolae 10D to high salinity levels of those found in Red Sea waters and conducted further modeled cultivations in nutrient enriched local sea water. It was determined that salt-water adapted C. merolae 10D could be cultivated with reduced nutrient inputs in local conditions. The results presented here indicate this may be a promising alternative species for algal bioprocesses in outdoor conditions in extreme desert summer environments.

Auxenochlorella sp. (strain UTEX 250-A) is a fast-growing, oleaginous alga that is an emerging reference organism for basic research, with broad biotechnological applications. Advancing UTEX 250-A as a biotechnological workhorse requires a deeper understanding of its nutritional demands, particularly under different trophic conditions. However, little is known about the specific acclimations that allow UTEX 250-A to thrive when essential nutrients are scarce. Here, we describe the formulation of a defined growth medium that supports the cultivation of UTEX 250-A with controlled trace levels of the essential micronutrients iron, copper, and zinc. Special attention was given to ensure that the medium was compatible with inductively coupled plasma mass spectrometry (ICP-MS) analysis, enabling the detection and minimization of metal contamination in the nanomolar range. The medium was designed to provide sufficient nutrients to support the mixotrophic growth of UTEX 250-A from an initial density of 105 cells/mL to a stationary density of 2.4x108 cells/mL, with additional nutrients supplied to accommodate metabolic and trophic transitions during stationary phase, when photosynthesis is restored due to carbon limitation. This replete medium provides a foundation for robust nutrient limitation studies in Auxenochlorella sp. (UTEX 250-A).

The use of cyanobacteria as bio-factories for production of numerous compounds of interest (biofuels, bioplastics ...) has attracted lots of attention mainly due to their simple nutritional requirements, coupled with the decrease in atmospheric CO2 levels. However, although cyanobacteria are easily genetically manipulated, there are few genetic tools developed and, in some cases, the modifications necessary for metabolic engineering are limited for this reason. We have developed a new positive selection marker based on the arsenic resistance system of the cyanobacterium Synechocystis sp. PCC 6803. In this cyanobacterium, resistance to arsenic is mediated by the arsBHC operon in which arsB encodes an arsenite transporter whose mutation confers hypersensitivity to the presence of both arsenite and arsenate. Using arsB mutant strain (SARSB) as recipient we introduced plasmids containing both arsB and an antibiotic resistance gene and transformants were selected using either arsenic or the antibiotic with similar efficiency. The plasmids and conditions to use the arsB gene as a selectable marker have been optimized. Furthermore, we have generated an integrative vector to delete the whole arsBHC operon that allows easy introduction of regulated genes in this locus. Analysis of this strain have shown that the{Delta} arsBHC mutant has a higher sensitivity to arsenite than the SARSB strain, even when they are complemented with an arsB copy. These suggest that arsH, arsC or both could have an additional role in arsenite resistance.

BackgroundThe axenisation of phototrophic eukaryotic microalgae has been studied for over a century, with antibiotics commonly employed to achieve axenic cultures. However, this approach often yields inconsistent outcomes and may contribute to the emergence of antibiotic-resistant microbes. A comprehensive review of microalgal species and the methods used to achieve axeny could provide insights into potentially effective workflows and identify gaps for future exploration. MethodsScholarly databases were systematically searched, supplemented by citation network analysis and AI-assisted tools, to collect studies on achieving axenic phototrophic eukaryotic microalgae cultures. Data about microalgal species, axenisation workflows, outcomes, and related factors (e.g., sampling locations, axenisation confirmation methods) were summarised. Network component analysis was used to identify clusters of commonly reported methods for diatoms, dinoflagellates, and green algae. A scoring framework was developed to assess the quality and reliability of evidence presented in the studies. ResultsPromising workflows circumventing the use of antibiotics appear to be filtration {leftrightarrow} washing {leftrightarrow} micropicking for diatoms, micropicking {leftrightarrow} subculturing {leftrightarrow} flow cytometry for dinoflagellates, and anoxy {leftrightarrow} photosensitisation {leftrightarrow} streak plating for green algae. Evidence from the literature indicates that a combination of microscopy (e.g., epifluorescence), cell counting (e.g., agar plating), and sequencing (16S and/or 18S) could enhance confidence in confirming axeny. ConclusionMore systematic and high quality primary research is required to identify effective workflows for other microalgal divisions and fortify / contradict the ones proposed herein based on network component analysis.

Microalgal productivity in mass cultures is limited by the inefficiency with which available light energy is utilized. In dense cultures, cells closest to the light source absorb more light energy than they can use and dissipate the excess, while light penetrance into the culture is steeply attenuated. Reducing microalgal light harvesting and/or dissipating capacity per cell may improve total light utilization efficiency in mass cultures. In this study, two transgenic lines of the diatom Thalassiosira pseudonana with altered photosynthetic pigment content are evaluated with respect to photosynthetic parameters, growth, and macromolecule accumulation. In one line, violaxanthin de-epoxidase-like 2 (VDL2) is overexpressed (OE), resulting in a reduction of the diadinoxanthin cycle pigments, which are involved in light energy dissipation (non-photochemical quenching, NPQ), accompanied by a stoichiometric increase in the light-harvesting pigment fucoxanthin. No differences in the maximum potential quantum yield of photosystem II (Fv/Fm) or light-limited photosynthetic rate () were found. However, when adapted to 30 {micro}mol photons m-2 sec-1, the VDL2 OE maximum relative electron transport rate (rETRmax) upon exposure to saturating light intensities was 86-95% of wild type (WT). When adapted to 300 {micro}mol photons m-2 sec-1, VDL2 OE saturated photosynthesis at 62-71% of the light intensity needed to saturate WT (Ek). NPQ was substantially lower at and below 300 {micro}mol photons m-2 sec-1. VDL2 OE accumulated up to 3.4 times as much triacylglycerol (TAG) as WT during exponential growth, and up to twice as much protein. Growth in terms of culture density was up to 7% slower. TAG and protein accumulation inversely correlated with NPQ. The second line evaluated was obtained by using antisense RNA to simultaneously silence or knock down (KD) both LUT1-like (LTL) genes, hypothesized to catalyze an intermediate carotenoid biosynthesis step of converting {beta}-carotene to zeaxanthin. Overall reduction of photosynthetic pigment content without altering the relative abundance of individual pigments resulted. No significant differences in photosynthetic parameters compared to WT were found. LTL KD grew at a rate comparable to WT and accumulated up to 40% more TAG during exponential growth, while protein content was reduced by 11-19%. LTL KD cells were elongated and 5-10% smaller than WT, and cultures contained auxospores, indicating stress that may relate to a cell cycle progression defect.

BackgroundMicroalgae are an important feedstock for the production of a wide variety of products, including biodiesel. Biodiesel, composed of fatty acid alkyl esters, is produced through the transesterification reaction of triacylglycerol (TAG). Microalgae store their energy reserves primarily as starch and TAGs. Therefore, several studies have focused on understanding the partitioning of carbon precursors between starch and TAG biosynthetic pathways. In this study, 5 starch mutants of Chlorella vulgaris were developed and cultured on different culture media. ResultsChlorella vulgaris starch mutants were generated through UV-random mutagenesis. Five starch mutants were selected for this study: four low-starch producing mutants (st27, st29, st43 and st54) and one high-starch producing mutant (st80). The starch mutants were cultured on media with different organic carbon sources, and lipid and biomass productivity were measured. Mixotrophic growth on glucose resulted in the highest lipid productivity in all the mutants, including st80, without compromising growth, whereas photoautotrophic growth generally did not result in changes in lipid productivity of the starch mutants. The highest increase in lipid productivity was observed for st27, with a 3.8-fold higher lipid productivity than wildtype. ConclusionsAll starch mutants increased their lipid productivities when grown mixotrophically on glucose, suggesting the overflow hypothesis could explain the partitioning of carbon between starch and TAGs. Out of the mutants generated in this work, st27 resulted in the highest increases in lipid productivities, reaching an increase of 380% when grown mixotrophically on glucose, without compromising growth. The high-starch producing mutant st80 provides insight into a possibility to develop starch- and TAG-rich microalgal biomass.

Cyanobacteria are promising microbial hosts for production of various industrially relevant compounds, such as succinate, a central metabolite of the tricarboxylic acid cycle (TCA). Cyanobacteria have been engineered to produce succinate during photoautotrophic growth, and are also able to secrete it during anoxic fermentation conditions. It has been assumed that under anoxic darkness, succinate can be formed by reduction of fumarate catalyzed by the succinate dehydrogenase complex (SDH), however, no characterization of SDH regarding this activity has been performed. In this study, we address this issue by generating strains of the unicellular cyanobacterium Synechocystis PCC 6803 (Synechocystis) deficient in one or several subunits of SDH, and investigating succinate accumulation in these strains during dark anaerobic fermentation. The results showed higher succinate accumulation in SDH deletion strains than in the wild type, indicating a succinate dehydrogenase activity of SDH rather than fumarate reduction under these conditions. We further explored the possibility of another potential route for succinate formation from fumarate via L-aspartate oxidase (Laspo). The gene encoding Laspo in Synechocystis could not be inactivated, indicating an essential function for this enzyme. Using purified SynLaspo, we could demonstrate in vitro that in addition to L-aspartate oxidation the enzyme exhibits an L-aspartate-fumarate oxidoreductase activity. We therefore suggest that reduction of fumarate to succinate during anoxic darkness can be a byproduct of the Laspo reaction, which is the first step in biosynthesis of NAD cofactors. This work contributes to the understanding of cyanobacterial TCA cycle for future engineering and sustainable production of dicarboxylic acids.

Stentor is a genus of large trumpet-shaped unicellular organisms in the ciliate phylum. Classically they have been used as models of cellular morphogenesis due to their large size and ability to regenerate, but some Stentor species have features that make them useful models for other types of studies as well. Stentor polymorphus is a widely distributed species that harbors green algal endosymbionts from the Chlorella genus. While interesting phenomenology in this species has been described, molecular tools have never been developed in this system. As technology has advanced, the use of emerging models like S. polymorphus has become more prevalent, and recently a set of transcriptomes for S. polymorphus was published. However, there are still technical hurdles to using S. polymorphus as an effective experimental system in the lab. Here I describe the identification and culture of a S. polymorphus population from North Carolina as well as the identification and cloning of homologs of -tubulin and the morphogenesis gene mob-1. Additionally, I demonstrate that RNA interference (RNAi) by feeding is effective against both of these homologs in S. polymorphus. The phenotypes observed in S. polymorphus were similar to phenotypes previously validated in S. coeruleus, a related Stentor species. A direct comparison of feeding RNAi between the two species revealed that RNAi appeared to be less effective in S. polymorphus. The ability to perform RNAi in S. polymorphus strengthens its use as an emerging model for exploring mechanisms of unicellular morphogenesis and regeneration or host-symbiont interactions and suggests that RNAi by bacterial feeding might be more broadly effective across the Stentor genus.

This paper evaluated and compared the relative microalgal biomass accumulation of rocking, floating, and stationary bag photobioreactors. Microalga Neochloris oleoabundans was grown in these photobioreactors in batch mode for 24 days under illumination. The 50 L plastic bags (cell suspension volume 25 L) were placed on the surface of a rocking platform, an artificial pond or a stationary platform. In the pond, waves were generated by electrical fans which shake and mix microalgal cells within the plastic bags. The bags were supplied with 5% CO2 in air under elevated pressure inside of the bags. The rocking bag method significantly increased biomass yields to approximately 3-4 g * L-1, as compared to 0.16 g * L-1 in the floating photobioreactor and only 0.03 g * L-1 in the stationary type photobioreactor.

BackgroundStentor, the genus of large trumpet-shaped ciliates, is well-known for its complex morphology and striking behaviors. Members of this genus are distributed throughout the world in a wide and diverse pool of freshwater ecosystems. Recently, the molecular phylogeny of Stentor has been explored through comparison of 18S small subunit (SSU) ribosomal DNA (rDNA) sequences, clarifying several previously mischaracterized species and species complexes. However, despite their wide distribution, to-date, only about a dozen species of Stentor have been described and verified by phylogenetic means. ResultsHere, we introduce the discovery of a new species within genus Stentor: Stentor stipatus spec. nov., so named for their distinctive cytosolic dark pigmented granules which surround the macronucleus and are also present cortically alongside cortically-distributed green microalgae. We present morphological, phylogenetic, ecological, and behavioral characterizations of these cells. Phylogenetic analysis of S. stipatus spec. nov. by comparison of SSU rDNA sequence suggests it is a distinct species from its closest relative, S. amethystinus. We demonstrate that S. stipatus spec. nov. is capable of habituation in response to repeated mechanical stimulation. Further, S. stipatus spec. nov. exhibits strongly directed positive phototaxis, like its relative S. pyriformis, but with a distinct action spectrum from both S. coeruleus and S. pyriformis. Finally, S. stipatus phototaxis response strength varies in a consistent pattern throughout the day, providing evidence of potential circadian regulation. ConclusionsThis work expands the current understanding of the ecological distribution of and behavioral features present within genus Stentor.

Target of rapamycin (TOR) is a conserved protein kinase that regulates the balance between catabolic and anabolic processes in response to nutrient availability. Although the central role of TOR kinase in nutrient stress responses is well-recognized, little is known about the molecular basis of TOR signaling in ecologically important secondary algae with plastids of red algal origin, such as diatoms, as assessing in vivo TOR kinase activity is a difficult task. To assess TOR kinase activity, the phosphorylation status of downstream components, such as ribosomal protein S6 (RPS6), must be measured. Unlike for model organisms, an antibody that detects phosphorylated (P-) RPS6 in diatoms is not commercially available. Therefore, we developed a convenient method in which P-RPS6 and non-P-RPS6 were detected via Phos-tag affinity electrophoresis and immunoblotting with a commercial antibody that cross-reacts with RPS6 (both P- and non-P-RPS6) in the diatom, Phaeodactylum tricornutum. Using this Phos-tag-based method, we observed a dose-dependent decrease in the P-RPS6/total RPS6 ratio in P. tricornutum cells treated with the TOR kinase inhibitor, AZD-8055. We also observed a reduction in the P-RPS6/total RPS6 ratio during the nitrogen-deficient culture of P. tricornutum, which indicated the inactivation of TOR kinase in response to nitrogen deficiency. Finally, we demonstrated the potential application of the Phos-tag-based method to other ecologically, evolutionarily, and industrially important secondary algae, such as Nannochloropsis oceanica (Stramenopiles), the haptophyte Tisochrysis lutea, and Euglena gracilis (Euglenid). As all experimental materials are commercially available, the Phos-tag-based method can be used to promote studies on TOR in diverse algae in different contexts.

PHB (poly-hydroxy-butyrate) represents a promising bioplastic variety with good biodegradation properties. Furthermore, PHB can be produced completely carbon-neutral when synthesized in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This model strain has a long history of various attempts to further boost its low amounts of produced intracellular PHB of ~15 % per cell-dry-weight (CDW). We have created a new strain that lacks the regulatory protein PirC (gene product of sll0944), which causes a rapid conversion of the intracellular glycogen pools to PHB under nutrient limiting conditions. To further improve the intracellular PHB content, two genes from the PHB metabolism, phaA and phaB from the known production strain Cupriavidus necator, were introduced under the regime of the strong promotor PpsbA2. The created strain, termed PPT1 ({Delta}sll0944-REphaAB), produced high amounts of PHB under continuous light as well under day-night rhythm. When grown in nitrogen and phosphor depleted medium, the cells produced up to 63 % / CDW. Upon the addition of acetate, the content was further increased to 81 % / CDW. The produced polymer consists of pure PHB, which is highly isotactic. The achieved amounts were the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.