Potential of Porous Substrate Bioreactors for Removal of Pollutants from Wastewater Using Microalgae.

Porous substrate bioreactors (PSBRs) are a new technology to grow microalgae immobilized in a dense culture and solve some problems linked to suspended cultivation. During recent years, this technology has been used in laboratory and pilot setups in different fields of environmental biotechnology, such as wastewater treatment. The aim of this short review is to introduce the PSBR technology, summarize the results obtained in removing some pollutants from wastewater, provide an assessment of the potential of PSBRs for wastewater treatment, and the subsequent use of the algal biomass for other purposes.

Plastid Genome Evolution of Two Colony-Forming Benthic Ochrosphaera neapolitana Strains (Coccolithales, Haptophyta).

Coccolithophores are well-known haptophytes that produce small calcium carbonate coccoliths, which in turn contribute to carbon sequestration in the marine environment. Despite their important ecological role, only two of eleven haptophyte plastid genomes are from coccolithophores, and those two belong to the order Isochrysidales. Here, we report the plastid genomes of two strains of Ochrosphaera neapolitana (Coccolithales) from Spain (CCAC 3688 B) and the USA (A15,280). The newly constructed plastid genomes are the largest in size (116,906 bp and 113,686 bp, respectively) among all the available haptophyte plastid genomes, primarily due to the increased intergenic regions. These two plastid genomes possess a conventional quadripartite structure with a long single copy and short single copy separated by two inverted ribosomal repeats. These two plastid genomes share 110 core genes, six rRNAs, and 29 tRNAs, but CCAC 3688 B has an additional CDS (ycf55) and one tRNA (trnL-UAG). Two large insertions at the intergenic regions (2 kb insertion between ycf35 and ycf45; 0.5 kb insertion in the middle of trnM and trnY) were detected in the strain CCAC 3688 B. We found the genes of light-independent protochlorophyllide oxidoreductase (chlB, chlN, and chlL), which convert protochlorophyllide to chlorophyllide during chlorophyll biosynthesis, in the plastid genomes of O. neapolitana as well as in other benthic Isochrysidales and Coccolithales species, putatively suggesting an evolutionary adaptation to benthic habitats.

Effect of LED Illumination Cycle and Carbon Sources on Biofilms of Haematococcus pluvialis in Pilot-Scale Angled Twin-Layer Porous Substrate Photobioreactors.

Light-emitting diodes are increasingly used as artificial light sources in Haematococcus pluvialis cultivation due to the fact of their energy advantages. The immobilized cultivation of H. pluvialis in pilot-scale angled twin-layer porous substrate photobioreactors (TL-PSBRs) was initially performed with a 14/10 h light/dark cycle and showed relatively low biomass growth and astaxanthin accumulation. In this study, the illumination time with red and blue LEDs at a light intensity of 120 µmol photons m-2 s-1 was increased to 16-24 h per day. With a light/dark cycle of 22/2 h, the biomass productivity of the algae was 7.5 g m-2 day-1, 2.4 times higher than in the 14/10 h cycle. The percentage of astaxanthin in the dry biomass was 2%, and the total amount of astaxanthin was 1.7 g m-2. Along with the increase in light duration, adding 10 or 20 mM NaHCO3 to the BG11-H culture medium over ten days of cultivation in angled TL-PSBRs did not increase the total amount of astaxanthin compared with only CO2 addition at a flow rate of 3.6 mg min-1 to the culture medium. Adding NaHCO3 with a 30-80 mM concentration inhibited algal growth and astaxanthin accumulation. However, adding 10-40 mM NaHCO3 caused algal cells to accumulate astaxanthin at a high percentage in dry weight after the first four days in TL-PSBRs.

New Angled Twin-layer Porous Substrate Photobioreactors for Cultivation of Nannochloropsis oculata.

An angled twin-layer porous substrate photobioreactor (TL-PSBR) using LED light was designed to cultivate Nannochloropsis oculata. Flocculation and sedimentation by modification of pH to 11 were determined as the optimal method for harvesting the N. oculata inoculum. The following optimised parameters were found: tilt angle 15°, Kraft 220 g m-2 paper as substrate material, initial inoculum density of 12.5 g m-2, 140 µmol photons m-2 s-1 light intensity, and a light/dark cycle of 6:6 (h). Test cultivation for 14 days was performed under optimised conditions. The total dried biomass standing crop was 75.5 g m-2 growth area with an average productivity of 6.3 g m-2 d-1, the productivity per volume of used culture medium was 126.2 mg/L d-1, total lipid content 21.9% (w/w), and the highest productivity of total lipids was 1.33 g m-2 d-1. The dry algal biomass contained 3% eicosapentaenoic acid (w/w), 3.7% palmitoleic acid (w/w), and 513 mg kg-1 vitamin E. The optimisation of N. oculata cultivation on an angled TL-PSBR system yielded promising results, and applications for commercial products need to be further explored.

Chromosome-level genome of Pedinomonas minor (Chlorophyta) unveils adaptations to abiotic stress in a rapidly fluctuating environment.

The Pedinophyceae (Viridiplantae) comprise a class of small uniflagellate algae with a pivotal position in the phylogeny of the Chlorophyta as the sister group of the 'core chlorophytes'. We present a chromosome-level genome assembly of the freshwater type species of the class, Pedinomonas minor. We sequenced the genome using Pacbio, Illumina and Hi-C technologies, performed comparative analyses of genome and gene family evolution, and analyzed the transcriptome under various abiotic stresses. Although the genome is relatively small (55 Mb), it shares many traits with core chlorophytes including number of introns and protein-coding genes, messenger RNA (mRNA) lengths, and abundance of transposable elements. Pedinomonas minor is only bounded by the plasma membrane, thriving in temporary habitats that frequently dry out. Gene family innovations and expansions and transcriptomic responses to abiotic stresses have shed light on adaptations of P. minor to its fluctuating environment. Horizontal gene transfers from bacteria and fungi have possibly contributed to the evolution of some of these traits. We identified a putative endogenization site of a nucleocytoplasmic large DNA virus and hypothesized that endogenous viral elements donated foreign genes to the host genome, their spread enhanced by transposable elements, located at gene boundaries in several of the expanded gene families.

Shared features and reciprocal complementation of the Chlamydomonas and Arabidopsis microbiota.

Microscopic algae release organic compounds to the region immediately surrounding their cells, known as the phycosphere, constituting a niche for colonization by heterotrophic bacteria. These bacteria take up algal photoassimilates and provide beneficial functions to their host, in a process that resembles the establishment of microbial communities associated with the roots and rhizospheres of land plants. Here, we characterize the microbiota of the model alga Chlamydomonas reinhardtii and reveal extensive taxonomic and functional overlap with the root microbiota of land plants. Using synthetic communities derived from C. reinhardtii and Arabidopsis thaliana, we show that phycosphere and root bacteria assemble into taxonomically similar communities on either host. We show that provision of diffusible metabolites is not sufficient for phycosphere community establishment, which additionally requires physical proximity to the host. Our data suggest the existence of shared ecological principles driving the assembly of the A. thaliana root and C. reinhardtii phycosphere microbiota, despite the vast evolutionary distance between these two photosynthetic organisms.

Lower Statistical Support with Larger Data Sets: Insights from the Ochrophyta Radiation.

It is commonly assumed that increasing the number of characters has the potential to resolve evolutionary radiations. Here, we studied photosynthetic stramenopiles (Ochrophyta) using alignments of heterogeneous origin mitochondrion, plastid, and nucleus. Surprisingly while statistical support for the relationships between the six major Ochrophyta lineages increases when comparing the mitochondrion (6,762 sites) and plastid (21,692 sites) trees, it decreases in the nuclear (209,105 sites) tree. Statistical support is not simply related to the data set size but also to the quantity of phylogenetic signal available at each position and our ability to extract it. Here, we show that this ability for current phylogenetic methods is limited, because conflicting results were obtained when varying taxon sampling. Even though the use of a better fitting model improved signal extraction and reduced the observed conflicts, the plastid data set provided higher statistical support for the ochrophyte radiation than the larger nucleus data set. We propose that the higher support observed in the plastid tree is due to an acceleration of the evolutionary rate in one short deep internal branch, implying that more phylogenetic signal per position is available to resolve the Ochrophyta radiation in the plastid than in the nuclear data set. Our work therefore suggests that, in order to resolve radiations, beyond the obvious use of data sets with more positions, we need to continue developing models of sequence evolution that better extract the phylogenetic signal and design methods to search for genes/characters that contain more signal specifically for short internal branches.

Cation and Anion Channelrhodopsins: Sequence Motifs and Taxonomic Distribution.

Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from four channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with a glutamate pattern identical to that in cryptophyte ACRs are cation selective. Desensitization is also broadly context dependent, as in one branch of stramenopile ACRs and their metagenomic homologs, its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, we identified two prasinophyte CCRs with red-shifted spectra to 585 nm. They exhibit a third residue pattern in their retinal-binding pockets distinctly different from those of the only two types of red-shifted channelrhodopsins known (i.e., the CCR Chrimson and RubyACRs). In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity. IMPORTANCE Channelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered prospective therapeutics, but their natural diversity and mechanisms remain poorly characterized. Genomic and metagenomic sequencing projects are producing an ever-increasing wealth of data, whereas biophysical characterization of the encoded proteins lags behind. In this study, we used manual and automated patch clamp recording of representative members of four channelrhodopsin families, including a family in dinoflagellates that we report in this study. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

Genome-wide analyses across Viridiplantae reveal the origin and diversification of small RNA pathway-related genes.

Small RNAs play a major role in the post-transcriptional regulation of gene expression in eukaryotes. Despite the evolutionary importance of streptophyte algae, knowledge on small RNAs in this group of green algae is almost non-existent. We used genome and transcriptome data of 34 algal and plant species, and performed genome-wide analyses of small RNA (miRNA & siRNA) biosynthetic and degradation pathways. The results suggest that Viridiplantae started to evolve plant-like miRNA biogenesis and degradation after the divergence of the Mesostigmatophyceae in the streptophyte algae. We identified two major evolutionary transitions in small RNA metabolism in streptophyte algae; during the first transition, the origin of DCL-New, DCL1, AGO1/5/10 and AGO4/6/9 in the last common ancestor of Klebsormidiophyceae and all other streptophytes could be linked to abiotic stress responses and evolution of multicellularity in streptophytes. During the second transition, the evolution of DCL 2,3,4, and AGO 2,3,7 as well as DRB1 in the last common ancestor of Zygnematophyceae and embryophytes, suggests their possible contribution to pathogen defense and antibacterial immunity. Overall, the origin and diversification of DICER and AGO along with several other small RNA pathway-related genes among streptophyte algae suggested progressive adaptations of streptophyte algae during evolution to a subaerial environment.

Giant DNA viruses make big strides in eukaryote evolution.

Nucleocytoplasmic large DNA viruses (NCLDVs) are widespread in the biosphere. This issue of Cell Host & Microbe, Nelson et al., and a recent Nature paper, Moniruzzaman et al., show NCLDVs can integrate into host genomes, highlighting a mechanism of large-scale virus-mediated horizontal gene transfer (vHGT) driving eukaryotic evolution.

The Draft Genome of Coelastrum proboscideum (Sphaeropleales, Chlorophyta).

Coelastrum proboscideum Bohlin, 1896 (Sphaeropleales, Scenedesmaceae, Chlorophyta) is a coenobial species with cosmopolitan distribution in diverse freshwater habitats. Coelastrum spp. are widely tested for biotechnological applications such as carotenoid and lipid production, and in bioremediation of wastewater. Here, we report the draft genome of C. proboscideum var. dilatatum strain SAG 217-2. The final assembly comprised 125,935,854 bp with over 8357 scaffolds. The whole-genome data is publicly available in the Nucleotide Sequence Archive (CNSA) of China National GeneBank (CNGB) (https://db.cngb.org/cnsa/) under the accession number CNA0014153.

Biomineralization Capacities of Chlorodendrophyceae: Correlation Between Chloroplast Morphology and the Distribution of Micropearls in the Cell.

Several species of the genus Tetraselmis (Chlorodendrophyceae, Chlorophyta) were recently discovered to possess unsuspected biomineralization capacities: they produce multiple intracellular inclusions of amorphous calcium carbonate (ACC), called micropearls. Early light-microscopists had spotted rows of refractive granules in some species, although without identifying their mineral nature. Scanning electron microscope (SEM) observations showed that the distribution of the micropearls in the cell forms a pattern, which appears to be characteristic for a given species. The present study shows that this pattern correlates with the shape of the chloroplast, which differs between Tetraselmis species, because micropearls align themselves along the incisions between chloroplast lobes. This was observed both by SEM and in live cells by light microscopy (LM) using Nomarski differential interference contrast. Additionally, molecular phylogenetic analyses, of rbcL and ITS2 gene sequences from diverse strains of Chlorodendrophyceae, corroborated the morphological observations by identifying two groups among nominal Tetraselmis spp. that differ in chloroplast morphology, micropearl arrangement, and ITS2 RNA secondary structure.

Comparison of Galdieria growth and photosynthetic activity in different culture systems.

In the last years, the acidothermophilic red microalga Galdieria sulphuraria has been increasingly studied for industrial applications such as wastewater treatment, recovery of rare earth elements, production of phycobilins. However, even now it is not possible an industrial cultivation of this organism because biotechnological research on G. sulphuraria and allied species is relatively recent and fragmented. Having in mind a possible scale-up for commercial applications, we have compared the growth and photosynthetic performance of G. sulphuraria in four suspended systems (Inclined bubble column, Decanter Laboratory Flask, Tubular Bioreactor, Ultra-flat plate bioreactor) and one immobilized system (Twin Layer Sytem). The results showed that G. sulphuraria had the highest growth, productivity and photosynthetic performance, when grown on the immobilized system, which also offers some economics advantages.

Author Correction: The genome of Prasinoderma coloniale unveils the existence of a third phylum within green plants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The genome of Prasinoderma coloniale unveils the existence of a third phylum within green plants.

Genome analysis of the pico-eukaryotic marine green alga Prasinoderma coloniale CCMP 1413 unveils the existence of a novel phylum within green plants (Viridiplantae), the Prasinodermophyta, which diverged before the split of Chlorophyta and Streptophyta. Structural features of the genome and gene family comparisons revealed an intermediate position of the P. coloniale genome (25.3 Mb) between the extremely compact, small genomes of picoplanktonic Mamiellophyceae (Chlorophyta) and the larger, more complex genomes of early-diverging streptophyte algae. Reconstruction of the minimal core genome of Viridiplantae allowed identification of an ancestral toolkit of transcription factors and flagellar proteins. Adaptations of P. coloniale to its deep-water, oligotrophic environment involved expansion of light-harvesting proteins, reduction of early light-induced proteins, evolution of a distinct type of C4 photosynthesis and carbon-concentrating mechanism, synthesis of the metal-complexing metabolite picolinic acid, and vitamin B1, B7 and B12 auxotrophy. The P. coloniale genome provides first insights into the dawn of green plant evolution.

Conductance Mechanisms of Rapidly Desensitizing Cation Channelrhodopsins from Cryptophyte Algae.

Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. "Bacteriorhodopsin-like" cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, the sequences of BCCR match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here, we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption, Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function.IMPORTANCE Cation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-visible (UV-vis) spectroscopy and photoelectrophysiology reveal that the desensitization is caused by rapid accumulation of a nonconductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

Rubisco and carbon-concentrating mechanism co-evolution across chlorophyte and streptophyte green algae.

Green algae expressing a carbon-concentrating mechanism (CCM) are usually associated with a Rubisco-containing micro-compartment, the pyrenoid. A link between the small subunit (SSU) of Rubisco and pyrenoid formation in Chlamydomonas reinhardtii has previously suggested that specific RbcS residues could explain pyrenoid occurrence in green algae. A phylogeny of RbcS was used to compare the protein sequence and CCM distribution across the green algae and positive selection in RbcS was estimated. For six streptophyte algae, Rubisco catalytic properties, affinity for CO2 uptake (K0.5 ), carbon isotope discrimination (δ13 C) and pyrenoid morphology were compared. The length of the ßA-ßB loop in RbcS provided a phylogenetic marker discriminating chlorophyte from streptophyte green algae. Rubisco kinetic properties in streptophyte algae have responded to the extent of inducible CCM activity, as indicated by changes in inorganic carbon uptake affinity, δ13 C and pyrenoid ultrastructure between high and low CO2 conditions for growth. We conclude that the Rubisco catalytic properties found in streptophyte algae have coevolved and reflect the strength of any CCM or degree of pyrenoid leakiness, and limitations to inorganic carbon in the aquatic habitat, whereas Rubisco in extant land plants reflects more recent selective pressures associated with improved diffusive supply of the terrestrial environment.