Autotrophic vs. Heterotrophic Cultivation of the Marine Diatom Cyclotella cryptica for EPA Production

Recently, the marketable value of ω-3 fatty acid, particularly eicosapentaenoic acid (EPA), increased considering their health effects for human consumption. Microalgae are considered a valuable and “green” source of EPA alternative to fish oils, but considerable efforts are necessary for their exploitation at an industrial level. Due to the high operation costs of photoautotrophic microalgae cultivation, heterotrophic growth represents a promising economic solution. Marine diatoms are the major ecological producers of ω-3 fatty acids. Few species of diatoms are capable to grow in the dark using organic carbon sources. The marine diatom Cyclotella cryptica was cultivated for 14 days under photoautotrophic and heterotrophic conditions to define the effects on growth parameters, lipid production, total fatty acids and EPA content. Photoautotrophic conditions led to a total EPA production of 1.6% of dry weight, 12.2 mg L−1 culture and productivity of 0.9 mg L−1 day−1. The heterotrophy cultures reported a total EPA production of 2.7% of dry cell weight, 18 mg L−1 culture, a productivity of 1.3 mg L−1 day−1, which are promising values in the prospective of improving culture parameters for the biotechnological exploitation of dark cultivation. C. cryptica could be a potential candidate for the heterotrophic production of EPA, also considering its robustness, capacity to resist to bacterial contaminations and plasticity of lipid metabolism.

DNP-supported solid-state NMR studies of 13C,15N,29Si-enriched biosilica of Cyclotella cryptica and Thalassiosira pseudonana

Solid-state NMR spectroscopy represents a powerful method for the investigation of diatom biosilica but detailed studies regarding its chemical composition and structural organization can be prohibited by insufficient spectroscopic sensitivity. Here, we used two-dimensional (2D) Dynamic Nuclear Polarization (DNP)-supported solid-state NMR experiments to obtain information about the molecular composition and supramolecular organization of proteins and carbohydrates in 13C,15N,29Si-labeled biosilica of C. cryptica. As a reference, we conducted DNP experiments on isotope-labeled biosilica of T. pseudonana. DNP-enhancement factors for different NMR signals, and thus, for different organic compounds, provide information about the supramolecular architecture of the biosilica. In addition, DNP-supported heteronuclear nitrogen-carbon correlation experiments allowed us to prove the presence of different structural elements of long chain polyamines (LCPAs) and revealed the occurrence of amine-nitrogen moieties exhibiting a correlation with carbonyl carbons that may indicate cross-linking of LCPAs to proteins as previously seen in studies on proteins extracted from other diatoms.

Solid-state NMR spectroscopic studies of 13C,15N,29Si-enriched biosilica from the marine diatom Cyclotella cryptica

Diatoms are algae producing micro- and nano-structured cell walls mainly containing amorphous silica. The shape and patterning of these cell walls is species-specific. Herein, the biosilica of Cyclotella cryptica, a centric marine diatom with a massive organic matrix, is studied. Solid-state NMR spectroscopy is applied to gain deeper insight into the interactions at the organic–inorganic interface of the cell walls. The various organic compounds like polysaccharides as well as proteins and long-chain polyamines (LCPAs) are detected by observation of heteronuclei like 13C and 15N whereas the silica phase is studied using 29Si NMR spectroscopy. The sensitivity of the NMR experiments is strongly enhanced by isotope-labeling of the diatoms during cultivation with 13C, 15N and 29Si. The presence of two different chitin species in the biosilica is demonstrated. This observation is supported by a monosaccharide analysis of the silica-associated organic matrix where a high amount of glucosamine is found. Moreover, the Rotational Echo Double Resonance (REDOR) experiment provides distance information for heteronuclear spins. 13C{29Si} REDOR experiments reveal proximities between different organic compounds and the silica phase. The closest contacts between silica and organic compounds appear for different signals in the 13C-chemical shift range of 40–60 ppm, the typical range for LCPAs.

Genome Sequences of Bacteria Associated with the Diatom Cyclotella cryptica Strain CCMP332

ABSTRACT We report draft genomes of two bacterial strains in the genera Hyphobacterium and Reichenbachiella, which are associated with the diatom Cyclotella cryptica strain CCMP332. Genomes from these strains were 2,691,501 bp and 3,325,829 bp, respectively, and will be useful for understanding interactions between diatoms and bacteria.

Solid-state NMR spectroscopic studies of 13C,15N,29Si-enriched biosilica from the marine diatom Cyclotella cryptica

Diatoms are algae producing micro- and nano-structured cell walls mainly containing amorphous silica. The shape and patterning of these cell walls is species-specific. Herein, the biosilica of Cyclotella cryptica, a centric marine diatom with a massive organic matrix, is studied. Solid-state NMR spectroscopy is applied to gain deeper insight into the interactions at the organic-inorganic interface of the cell walls. The various organic compounds like polysaccharides as well as proteins and long-chain polyamines (LCPAs) are detected by observation of heteronuclei like 13C and 15N whereas the silica phase is studied using 29Si NMR spectroscopy. The sensitivity of the NMR experiments is strongly enhanced by isotope-labeling of the diatoms during cultivation with 13C, 15N and 29Si. The presence of two different chitin species in the biosilica is demonstrated. This observation is supported by a monosaccharide analysis of the silica-associated organic matrix where a high amount of glucosamine is found. Moreover, the Rotational Echo Double Resonance (REDOR) experiment provides distance information for heteronuclear spins. 13C{29Si} REDOR experiments reveal proximities between different organic compounds and the silica phase. The closest contacts between silica and organic compounds appear for different signals in the 13C-chemical shift range of 40 – 60 ppm, the typical range for LCPAs.

Improved Reference Genome for Cyclotella cryptica CCMP332, a Model for Cell Wall Morphogenesis, Salinity Adaptation, and Lipid Production in Diatoms (Bacillariophyta)

The diatom, Cyclotella cryptica, is a well-established model species for physiological studies and biotechnology applications of diatoms. To further facilitate its use as a model diatom, we report an improved reference genome assembly and annotation for C. cryptica strain CCMP332. We used a combination of long- and short-read sequencing to assemble a high-quality and contaminant-free genome. The genome is 171 Mb in size and consists of 662 scaffolds with a scaffold N50 of 494 kb. This represents a 176-fold decrease in scaffold number and 41-fold increase in scaffold N50 compared to the previous assembly. The genome contains 21,250 predicted genes, 75% of which were assigned putative functions. Repetitive DNA comprises 59% of the genome, and an improved classification of repetitive elements indicated that a historically steady accumulation of transposable elements has contributed to the relatively large size of the C. cryptica genome. The high-quality C. cryptica genome will serve as a valuable reference for ecological, genetic, and biotechnology studies of diatoms.

DNP-Supported Solid-State NMR Studies of 13C,15N,29Si-Enriched Biosilica of Cyclotella Cryptica and Thalassiosira Pseudonana

Solid-state NMR spectroscopy represents a powerful method for the investigation of diatom biosilica but detailed studies regarding its chemical composition and structural organization can be prohibited by insufficient spectroscopic sensitivity. Here, we used two-dimensional (2D) Dynamic Nuclear Polarization (DNP)-supported solid-state NMR experiments to obtain information about the molecular composition and supramolecular organization of proteins and carbohydrates in13C,15N,29Si-labeled biosilica of C. cryptica. As a reference, we conducted DNP experiments on isotope-labeled biosilica of T. pseudonana. DNP-enhancement factors for different NMR signals, and thus, for different organic compounds, provide information about the supramolecular architecture of the biosilica. In addition, DNP-supported heteronuclear nitrogen-carbon correlation experiments allowed us to prove the presence of different LCPAs (long chain polyamines) and revealed the occurrence of amine-nitrogen moieties exhibiting a correlation with carbonyl carbons that may indicate cross-linking of LCPAs to proteins as previously seen in studies on proteins extracted from other diatoms.

Solid-state NMR spectroscopic studies of 13C,15N,29Si-enriched biosilica from the marine diatom Cyclotella cryptica

Diatoms are algae producing micro- and nano-structured cell walls mainly containing amorphous silica. The shape and patterning of these cell walls is species-specific. Herein, the biosilica of Cyclotella cryptica, a centric marine diatom with a massive organic matrix, is studied. Solid-state NMR spectroscopy is applied to gain deeper insight into the interactions at the organic-inorganic interface of the cell walls. The various organic compounds like polysaccharides as well as proteins and long-chain polyamines (LCPAs) are detected by observation of heteronuclei like 13C and 15N whereas the silica phase is studied using 29Si NMR spectroscopy. The sensitivity of the NMR experiments is strongly enhanced by isotope-labeling of the diatoms during cultivation with 13C, 15N and 29Si. The presence of two different chitin species in the biosilica is demonstrated. This observation is supported by a monosaccharide analysis of the silica-associated organic matrix where a high amount of glucosamine is found. Moreover, the Rotational Echo Double Resonance (REDOR) experiment provides distance information for heteronuclear spins. 13C{29Si} REDOR experiments reveal proximities between different organic compounds and the silica phase. The closest contacts between silica and organic compounds appear for different signals in the 13C-chemical shift range of 40–60 ppm, the typical range for LCPAs.

Improved Reference Genome for Cyclotella Cryptica CCMP332, a Model for Cell Wall Morphogenesis, Salinity Adaptation, and Lipid Production in Diatoms (Bacillariophyta)

ABSTRACTThe diatom, Cyclotella cryptica, is a well-established experimental model for physiological studies and, more recently, biotechnology applications of diatoms. To further facilitate its use as a model diatom species, we report an improved reference genome assembly and annotation for C. cryptica strain CCMP332. We used a combination of long- and short-read sequencing to assemble a high-quality and contaminant-free genome. The genome is 171 Mb in size and consists of 662 scaffolds with a scaffold N50 of 494 kb. This represents a 176-fold decrease in scaffold number and 41-fold increase in scaffold N50 compared to the previous assembly. The genome contains 21,250 predicted genes, 75% of which were assigned putative functions. Repetitive DNA comprises 59% of the genome, and an improved classification of repetitive elements indicated that a historically steady accumulation of transposable elements has contributed to the relatively large size of the C. cryptica genome. The high-quality C. cryptica genome will serve as a valuable reference for ecological, genetic, and biotechnology studies of diatoms.Data available fromNCBI BioProjects PRJNA628076 and PRJNA589195