Metabolomics and lipid profile analysis of Coccomyxa melkonianii SCCA 048.

With an unsupervised GC-MS metabolomics approach, polar metabolite changes of the microalgae Coccomyxa melkonianii SCCA 048 grown under standard conditions for seven weeks were studied. C. melkonianii was sampled at the Rio Irvi River, in the mining site of Montevecchio-Ingurtosu (Sardinia, Italy), which is severely contaminated by heavy metals and shows high concentrations of sulfates. The partial-least-square (PLS) analysis of the GC-MS data indicated that growth of C. melkonianii was characterized by an increase of the levels of threonic acid, myo-inositol, malic acid, and fumaric acid. Furthermore, at the sixth week of exponential phase the lipid fingerprint of C. melkonianii was studied by LC-QTOF-MS. C. melkonianii lipid extract characterized through an iterative MS/MS analysis showed the following percent levels: 61.34 ± 0.60% for triacylglycerols (TAG); 11.55 ± 0.09% for diacylglyceryltrimethyl homoserines (DGTS), 11.34 ± 0.10% for sulfoquinovosyldiacylglycerols (SQDG) and, 5.29 ± 0.04% for lysodiacylglyceryltrimethyl homoserines (LDGTS). Noteworthy, we were able to annotate different fatty acid ester of hydroxyl fatty acid, such as FAHFA (18:1_20:3), FAHFA (18:2_20:4), FAHFA (18:0_20:2), and FAHFA (18:1_18:0), with relevant biological activity. These approaches can be useful to study the biochemistry of this extremophile algae in the view of its potential exploitation in the phycoremediation of polluted mining areas.

Suitability of the thawed algae for transmission electron microscopy study: Ultrastructural investigation on Coccomyxa melkonianii SCCA 048.

Morphological and ultrastructural investigations are crucial for the identification and characterization of species such as microalgae, microorganisms that greatly change their morphology and physiology during their life cycle. Transmission electron microscopy (TEM) is an excellent tool for the ultrastructural observation of cells and their components. To date, limited ultrastructural studies have been carried out on microalgae, due to the difficulties in sample preparation. The aim of this work is to establish an appropriate fixation method that allows to better preserve the algal ultrastructure and test the suitability of the thawed algae for TEM observation. Fresh and thawed algae (Coccomyxa melkonianii SCCA 048) were fixed with different TEM fixation methods (a mix of glutaraldehyde and paraformaldehyde for several incubation times, sometimes preceded by a prefixation in cold methanol). The ultrastructural images obtained from fresh algae were compared to those obtained from frozen biomass. The best morphological results were achieved by fixing fresh algae in 1% paraformaldehyde and 1.25% glutaraldehyde for 5 hr. Pretreating with frozen methyl alcohol reduced fixation time to 2 hr. Both fresh and frozen algae ultrastructure were rather well preserved also with 1% paraformaldehyde and 1.25% glutaraldehyde for 2 hr. Ultrastuctural morphological images of the thawed algae demonstrated that also frozen samples can be used in TEM research, widening specimen suitability by means of this technique.

Extremophile Microalgae: the potential for biotechnological application.

Microalgae are photosynthetic microorganisms that use sunlight as an energy source, and convert water, carbon dioxide, and inorganic salts into algal biomass. The isolation and selection of microalgae, which allow one to obtain large amounts of biomass and valuable compounds, is a prerequisite for their successful industrial production. This work provides an overview of extremophile algae, where their ability to grow under harsh conditions and the corresponding accumulation of metabolites are addressed. Emphasis is placed on the high-value products of some prominent algae. Moreover, the most recent applications of these microorganisms and their potential exploitation in the context of astrobiology are taken into account.

Behavior of the extremophile green alga Coccomyxa melkonianii SCCA 048 in terms of lipids production and morphology at different pH values.

The extremophile green alga Coccomyxa melkonianii SCCA 048 was investigated to evaluate its ability to grow in culture media with different pH. Specifically, Coccomyxa melkonianii was sampled in the Rio Irvi river (Sardinia, Italy) which is severely polluted by heavy metals as a result of abandoned mining activities. In this study, the strain was cultivated in growth media where the pH was kept fixed at the values of 4.0, 6.8 and 8.0, respectively. During the investigation, a significant phenotypic plasticity of this strain was observed. The strain grew well in the pH range 4.0-8.0, while the optimal value for its growth was 6.8. Furthermore, maximum lipid contents of about 24 and 22 %wt were achieved at the end of cultivation when using pH 4.0 and 8.0, respectively. Finally, the analysis of fatty acid methyl esters (FAMEs) highlights the presence of suitable amounts of compounds which can be profitably exploited in the food, nutraceutical, and cosmetic industry. This aspect, coupled with the possibility of cultivating Coccomyxa melkonianii under extreme pH conditions in economic open ponds, makes this strain an interesting candidate for several biotechnological applications.

Molecular phylogeny, systematics, and revision of the type species of Lobomonas, L. francei (Volvocales, Chlorophyta) and closely related taxa.

In the present study, three new strains of the rare volvocalean green alga Lobomonas were isolated from field-collected samples, one from Sardinia (Italy) and two from Argentina, and comparatively studied. The Sardinian and one of the Argentinian strains were identified as Lobomonas francei, the type species of the genus, whereas the second Argentinian strain corresponded to L. panduriformis. Two additional nominal species of Lobomonas from culture collections (L. rostrata and L. sphaerica) were included in the analysis and shown to be morphologically and molecularly identical to the L. francei strains. The presence, number, and shapes of cell wall lobes, the diagnostic criterion of Lobomonas, were shown to be highly variable depending on the chemical composition of the culture medium used. The analyses by SEM gave evidence that the cell wall lobes in Lobomonas originate at the junctions of adjacent cell wall plates by extrusion of gelatinous material. The four L. francei strains had identical nrRNA gene sequences and differed by only one or two substitutions in the ITS1 + ITS2 sequences. In the phylogenetic analyses, L. francei and L. panduriformis were sister taxa; however, another nominal Lobomonas species (L. monstruosa) did not belong to this genus. Lobomonas, together with taxa designated as Vitreochlamys, Tetraspora, and Paulschulzia, formed a monophyletic group that in the combined analyses was sister to the "Chlamydomonas/Volvox-clade." Based on these results, Lobomonas was revised, the diagnosis of the type species emended, a lectotype and an epitype designated, and several taxa synonymized with the type species.

Autotrophic growth and lipid production of Chlorella sorokiniana in lab batch and BIOCOIL photobioreactors: Experiments and modeling.

A novel mathematical model for the quantitative assessment of the effect of dissolved nitrogen on the autotrophic batch-growth and lipid accumulation of Chlorella sorokiniana, is proposed in this work. Model results have been validated through comparison with suitable experimental data performed in lab photobioreactors. Further experiments have been then performed using the BIOCOIL photobioreactor operated in fed-batch mode. The experimental results, which show that a maximum growth rate of 0.52day(-1) and a lipid content equal to 25%wt can be achieved with the BIOICOIL, have been successfully predicted through the proposed model. Therefore, the model might represent a first step toward the development of a tool for the scale-up and optimization of the operating conditions of BIOCOIL photobioreactors. Finally, the fatty acid methyl esters obtained by trans-esterification of lipids extracted from C. sorokiniana, have been analyzed in view of the assessment of their usability for producing biodiesel.

DNA-Based Taxonomy in Ecologically Versatile Microalgae: A Re-Evaluation of the Species Concept within the Coccoid Green Algal Genus Coccomyxa (Trebouxiophyceae, Chlorophyta).

Coccomyxa is a genus of unicellular green algae of the class Trebouxiophyceae, well known for its cosmopolitan distribution and great ecological amplitude. The taxonomy of this genus has long been problematic, due to reliance on badly-defined and environmentally variable morphological characters. In this study, based on the discovery of a new species from an extreme habitat, we reassess species circumscription in Coccomyxa, a unicellular genus of the class Trebouxiophyceae, using a combination of ecological and DNA sequence data (analyzed with three different methods of algorithmic species delineation). Our results are compared with those of a recent integrative study of Darienko and colleagues that reassessed the taxonomy of Coccomyxa, recognizing 7 species in the genus. Expanding the dataset from 43 to 61 sequences (SSU + ITS rDNA) resulted in a different delimitation, supporting the recognition of a higher number of species (24 to 27 depending on the analysis used, with the 27-species scenario receiving the strongest support). Among these, C. melkonianii sp. nov. is described from material isolated from a river highly polluted by heavy metals (Rio Irvi, Sardinia, Italy). Analyses performed on ecological characters detected a significant phylogenetic signal in six different characters. We conclude that the 27-species scenario is presently the most realistic for Coccomyxa and we suggest that well-supported lineages distinguishable by ecological preferences should be recognized as different species in this genus. We also recommend that for microbial lineages in which the overall diversity is unknown and taxon sampling is sparse, as is often the case for green microalgae, the results of analyses for algorithmic DNA-based species delimitation should be interpreted with extreme caution.

Can Artificial Ecosystems Enhance Local Biodiversity? The Case of a Constructed Wetland in a Mediterranean Urban Context.

Constructed wetlands (CW) are considered a successful tool to treat wastewater in many countries: their success is mainly assessed observing the rate of pollution reduction, but CW can also contribute to the conservation of ecosystem services. Among the many ecosystem services provided, the biodiversity of CW has received less attention. The EcoSistema Filtro (ESF) of the Molentargius-Saline Regional Natural Park is a constructed wetland situated in Sardinia (Italy), built to filter treated wastewater, increase habitat diversity, and enhance local biodiversity. A floristic survey has been carried out yearly 1 year after the construction of the artificial ecosystem in 2004, observing the modification of the vascular flora composition in time. The flora of the ESF accounted for 54% of the whole Regional Park's flora; alien species amount to 12%; taxa of conservation concern are 6%. Comparing the data in the years, except for the biennium 2006/2007, we observed a continuous increase of species richness, together with an increase of endemics, species of conservation concern, and alien species too. Once the endemics appeared, they remained part of the flora, showing a good persistence in the artificial wetland. Included in a natural park, but trapped in a sprawling and fast growing urban context, this artificial ecosystem provides multiple uses, by preserving and enhancing biodiversity. This is particularly relevant considering that biodiversity can act as a driver of sustainable development in urban areas where most of the world's population lives and comes into direct contact with nature.

Complete genome sequence of mitochondrial DNA (mtDNA) of Chlorella sorokiniana.

The complete sequence of mitochondrial genome of the Chlorella sorokiniana strain (SAG 111-8 k) is presented in this work. Within the Chlorella genus, it represents the second species with a complete sequenced and annotated mitochondrial genome (GenBank accession no. KM241869). The genome consists of circular chromosomes of 52,528 bp and encodes a total of 31 protein coding genes, 3 rRNAs and 26 tRNAs. The overall AT contents of the C. sorokiniana mtDNA is 70.89%, while the coding sequence is of 97.4%.

Complete genome sequence of chloroplast DNA (cpDNA) of Chlorella sorokiniana.

The complete chloroplast genome sequence of Chlorella sorokiniana strain (SAG 111-8 k) is presented in this study. The genome consists of circular chromosomes of 109,811 bp, which encode a total of 109 genes, including 74 proteins, 3 rRNAs and 31 tRNAs. Moreover, introns are not detected and all genes are present in single copy. The overall AT contents of the C. sorokiniana cpDNA is 65.9%, the coding sequence is 59.1% and a large inverted repeat (IR) is not observed.

Complete sequence and characterization of mitochondrial and chloroplast genome of Chlorella variabilis NC64A.

The complete nucleotide sequences of the mitochondrial (mtDNA) and chloroplast (cpDNA) genomes of Chlorella variabilis NC64A (Trebouxiophyceae) have been determined in this study (GenBank accession no. KP271968 and KP271969, respectively). The mt genome assembles as a circle of 78,500 bp and contains 62 genes, including 32 protein-coding, 27 tRNA and 3 rRNA genes. The overall GC content is 28.2%, while the coding sequence is 34%. The cp genome forms a circle of 124,793 bp, containing 114 genes, including 79 protein-coding, 32 tRNA and 3 rRNA genes. The overall GC content is 33,9%, while the coding sequence is 50%.