Indigenous microalgae strains characterization for a sustainable biodiesel production.

Microalgae have been widely recognized as a promising feedstock for sustainable biofuels production to tackle global warming and pollution issues related to fossil fuels uses. This study identified and analyzed indigenous microalgae strains for biodiesel production, specifically Chlorella vulgaris and Coelastrella thermophila var. globulina, from two distinct locations in Algeria. Molecular identification confirmed their identity, and the microalgae exhibited notable growth characteristics. Local Chlorella vulgaris and Coelastrella thermophila var. globulina showed good growth and high biomass yield, compared to Chlorella vulgaris CCAP211/11B reaching a weight of 1.48 g L-1 , 1.95 g L-1 , and 2.10 g L-1 , respectively. Lipids content of local Chlorella vulgaris, Coelastrella thermophila var. globulina, and Chlorella vulgaris CCAP211/11B, were found to be 31.39 ± 3.3%, 17 ± 2.26%, and 19 ± 0.64%, respectively. Chlorella vulgaris stood out as a candidate for biodiesel production due to its equilibrium between SFA and PUFA (43.24% and 45.27%). FAs are predominated by SFA and MUFA for Coelastrella thermophila var. globulina with value of 81.49% (SFA+MUFA). Predicted biodiesel qualities comply with ASTM6751 and EN14214 standards. Studied microalgae have therefore a promising potential for biodiesel production. However, optimising cultivation conditions is necessary to enhance biomass and lipids yield at a large scale.

Microalgae and Cyanobacteria Strains as Producers of Lipids with Antibacterial and Antibiofilm Activity.

Lipids are one of the primary metabolites of microalgae and cyanobacteria, which enrich their utility in the pharmaceutical, feed, cosmetic, and chemistry sectors. This work describes the isolation, structural elucidation, and the antibiotic and antibiofilm activities of diverse lipids produced by different microalgae and cyanobacteria strains from two European collections (ACOI and LEGE-CC). Three microalgae strains and one cyanobacteria strain were selected for their antibacterial and/or antibiofilm activity after the screening of about 600 strains carried out under the NoMorFilm European project. The total organic extracts were firstly fractionated using solid phase extraction methods, and the minimum inhibitory concentration and minimal biofilm inhibitory concentration against an array of human pathogens were determined. The isolation was carried out by bioassay-guided HPLC-DAD purification, and the structure of the isolated molecules responsible for the observed activities was determined by HPLC-HRESIMS and NMR methods. Sulfoquinovosyldiacylglycerol, monogalactosylmonoacylglycerol, sulfoquinovosylmonoacylglycerol, α-linolenic acid, hexadeca-4,7,10,13-tetraenoic acid (HDTA), palmitoleic acid, and lysophosphatidylcholine were found among the different active sub-fractions selected. In conclusion, cyanobacteria and microalgae produce a great variety of lipids with antibiotic and antibiofilm activity against the most important pathogens causing severe infections in humans. The use of these lipids in clinical treatments alone or in combination with antibiotics may provide an alternative to the current treatments.

Toward Modern Classification of Eustigmatophytes, Including the Description of Neomonodaceae Fam. Nov. and Three New Genera1.

The class Eustigmatophyceae includes mostly coccoid, freshwater algae, although some genera are common in terrestrial habitats and two are primarily marine. The formal classification of the class, developed decades ago, does not fit the diversity and phylogeny of the group as presently known and is in urgent need of revision. This study concerns a clade informally known as the Pseudellipsoidion group of the order Eustigmatales, which was initially known to comprise seven strains with oval to ellipsoidal cells, some bearing a stipe. We examined those strains as well as 10 new ones and obtained 18S rDNA and rbcL gene sequences. The results from phylogenetic analyses of the sequence data were integrated with morphological data of vegetative and motile cells. Monophyly of the Pseudellipsoidion group is supported in both 18S rDNA and rbcL trees. The group is formalized as the new family Neomonodaceae comprising, in addition to Pseudellipsoidion, three newly erected genera. By establishing Neomonodus gen. nov. (with type species Neomonodus ovalis comb. nov.), we finally resolve the intricate taxonomic history of a species originally described as Monodus ovalis and later moved to the genera Characiopsis and Pseudocharaciopsis. Characiopsiella gen. nov. (with the type species Characiopsiella minima comb. nov.) and Munda gen. nov. (with the type species Munda aquilonaris) are established to accommodate additional representatives of the polyphyletic genus Characiopsis. A morphological feature common to all examined Neomonodaceae is the absence of a pyrenoid in the chloroplasts, which discriminates them from other morphologically similar yet unrelated eustigmatophytes (including other Characiopsis-like species).

Chemotaxonomic Profiling Through NMR1.

A metabolite screening of cyanobacteria was performed by nuclear magnetic resonance (NMR) analysis of the soluble material obtained through sequential extraction of the biomass with three different extractive ability solvents (hexane, ethyl acetate, and methanol). Twenty-five strains from the Coimbra Collection of Algae (ACOI) belonging to different orders in the botanical code that represent three subsections of the Stainer-Rippka classification were used. The 1 H NMR spectra of hexane extracts showed that only two strains of Nostoc genus accumulated triacylglycerols. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols were the major components of the ethyl acetate extracts in a mono- to digalactosyldiacylglycerols ratio of 4.5 estimated by integration of the signals at δ 3.99 and 3.94 ppm (sn3 glycerol methylene). Oligosaccharides of sucrose and mycosporine-like amino acids, among other polar metabolites, were detected in the methanolic extracts. Strains of Nostocales order contained heterocyst glycolipids, whereas sulphoquinovosyldiacylglycerols were absent in one of the studied strains (Microchaete tenera ACOI 1451). Phosphathidylglycerol was identified as the major phospholipid in the methanolic extracts together with minor amounts of phosphatidylcholine based on 1 H, 31 P 2D correlation experiments. Chemotaxonomic information could be easily obtained through the analysis of the δ 3.0-0.5 ppm (fatty acid distribution) and δ 1.2-1.1 ppm (terminal methyl groups of the aglycons in heterocyst glycolipids) regions of the 1 H NMR spectra of the ethyl acetate and methanol extracts, respectively.

Inhibition of Bacterial and Fungal Biofilm Formation by 675 Extracts from Microalgae and Cyanobacteria.

Bacterial biofilms are complex biological systems that are difficult to eradicate at a medical, industrial, or environmental level. Biofilms confer bacteria protection against external factors and antimicrobial treatments. Taking into account that about 80% of human infections are caused by bacterial biofilms, the eradication of these structures is a great priority. Biofilms are resistant to old-generation antibiotics, which has led to the search for new antimicrobials from different sources, including deep oceans/seas. In this study, 675 extracts obtained from 225 cyanobacteria and microalgae species (11 phyla and 6 samples belonging to unknown group) were obtained from different culture collections: The Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC), the Coimbra Collection of Algae (ACOI) from Portugal, and the Roscoff Culture Collection (RCC) from France. The largest number of samples was made up of the microalgae phylum Chlorophyta (270) followed by Cyanobacteria (261). To obtain a large range of new bioactive compounds, a method involving three consecutive extractions (hexane, ethyl acetate, and methanol) was used. The antibiofilm activity of extracts was determined against seven different bacterial species and two Candida strains in terms of minimal biofilm inhibitory concentration (MBIC). The highest biofilm inhibition rates (%) were achieved against Candida albicans and Enterobacter cloacae. Charophyta, Chlorophyta, and Cyanobacteria were the most effective against all microorganisms. In particular, extracts of Cercozoa phylum presented the lowest MBIC50 and MBIC90 values for all the strains except C. albicans.

Plastid Genomes and Proteins Illuminate the Evolution of Eustigmatophyte Algae and Their Bacterial Endosymbionts.

Eustigmatophytes, a class of stramenopile algae (ochrophytes), include not only the extensively studied biotechnologically important genus Nannochloropsis but also a rapidly expanding diversity of lineages with much less well characterized biology. Recent discoveries have led to exciting additions to our knowledge about eustigmatophytes. Some proved to harbor bacterial endosymbionts representing a novel genus, Candidatus Phycorickettsia, and an operon of unclear function (ebo) obtained by horizontal gene transfer from the endosymbiont lineage was found in the plastid genomes of still other eustigmatophytes. To shed more light on the latter event, as well as to generally improve our understanding of the eustigmatophyte evolutionary history, we sequenced plastid genomes of seven phylogenetically diverse representatives (including new isolates representing undescribed taxa). A phylogenomic analysis of plastid genome-encoded proteins resolved the phylogenetic relationships among the main eustigmatophyte lineages and provided a framework for the interpretation of plastid gene gains and losses in the group. The ebo operon gain was inferred to have probably occurred within the order Eustigmatales, after the divergence of the two basalmost lineages (a newly discovered hitherto undescribed strain and the Pseudellipsoidion group). When looking for nuclear genes potentially compensating for plastid gene losses, we noticed a gene for a plastid-targeted acyl carrier protein that was apparently acquired by horizontal gene transfer from Phycorickettsia. The presence of this gene in all eustigmatophytes studied, including representatives of both principal clades (Eustigmatales and Goniochloridales), is a genetic footprint indicating that the eustigmatophyte-Phycorickettsia partnership started no later than in the last eustigmatophyte common ancestor.

A gene transfer event suggests a long-term partnership between eustigmatophyte algae and a novel lineage of endosymbiotic bacteria.

Rickettsiales are obligate intracellular bacteria originally found in metazoans, but more recently recognized as widespread endosymbionts of various protists. One genus was detected also in several green algae, but reports on rickettsialean endosymbionts in other algal groups are lacking. Here we show that several distantly related eustigmatophytes (coccoid algae belonging to Ochrophyta, Stramenopiles) are infected by Candidatus Phycorickettsia gen. nov., a new member of the family Rickettsiaceae. The genome sequence of Ca. Phycorickettsia trachydisci sp. nov., an endosymbiont of Trachydiscus minutus CCALA 838, revealed genomic features (size, GC content, number of genes) typical for other Rickettsiales, but some unusual aspects of the gene content were noted. Specifically, Phycorickettsia lacks genes for several components of the respiration chain, haem biosynthesis pathway, or c-di-GMP-based signalling. On the other hand, it uniquely harbours a six-gene operon of enigmatic function that we recently reported from plastid genomes of two distantly related eustigmatophytes and from various non-rickettsialean bacteria. Strikingly, the eustigmatophyte operon is closely related to the one from Phycorickettsia, suggesting a gene transfer event between the endosymbiont and host lineages in early eustigmatophyte evolution. We hypothesize an important role of the operon in the physiology of Phycorickettsia infection and a long-term eustigmatophyte-Phycorickettsia coexistence.

Is axenicity crucial to cryopreserve microalgae?

Large culture collections of microalgae and cyanobacteria such as the Coimbra Collection of Algae (ACOI) hold unialgal cultures consisting of a population of cells/colonies of a certain species. These cultures are usually non-axenic, as other organisms such as bacteria and microfungi are also present in culture due to co-isolation. Attention has been recently given to partner organisms since studies indicate that some bacteria are important for nutrient uptake of the algal cells, acting as simbionts. Despite this benign effect in the actively growing cultures, when cryopreservation is applied for inactive-stage storage, these organisms may recover faster than the algae, thus affecting their recovery and the viability assessments. In this study, a set of mucilaginous ACOI microalgae were selected, cell features known for their relevance in cryopreservation success were recorded and simple two-step cryopreservation tests were applied. Thawed samples were transferred to fresh culture medium for recovery. Viability was assessed and partner organism proliferation (pop) was recorded. Results were analyzed by t-tests. Statistical models allowed us to support the known tendency for small, unicellular algae with no outer structures to be successfully cryopreserved and the negative effect of vacuoles in the cell prior to cryopreservation. On average cryopreservation with MeOH or Me2SO led to the recovery of nearly half the cells. It was found that the cryoprotection step with MeOH is when pop is triggered and that the use of Me2SO can prevent this effect. Progress on understanding the cultured consortia will assist the improvement of cryopreservation and research using microalgal cultures.

Standard PREanalytical Codes: A New Paradigm for Environmental Biobanking Sectors Explored in Algal Culture Collections.

The Standard PREanalytical Code (SPREC) was developed by the medical/clinical biobanking sector motivated by the need to harmonize biospecimen traceability in preanalytical processes and enable interconnectivity and interoperability between different biobanks, research consortia, and infrastructures. The clinical SPREC (01) consists of standard preanalytical variable options (7-code elements), which comprise published and (ideally) validated methodologies. Although the SPREC has been designed to facilitate clinical research, the concept could have utility in biorepositories and culture collections that service environmental and biodiversity communities. The SPREC paradigm can be applied to different storage regimes across all types of biorepository. The objective of this article is to investigate adapting the code in nonclinical biobanks using algal culture collections and their cryostorage as a case study. The SPREC (01) is recalibrated as a putative code that might be adopted for biobanks holding different types of biodiversity; it is extended to include optional coding from the point of sample collection to postcryostorage manipulations, with the caveat that the processes are undertaken by biorepository personnel.

The use of physical and virtual infrastructures for the validation of algal cryopreservation methods in international culture collections.

Two cryopreservation methods, colligative cryoprotection coupled with controlled cooling and vitrification-based, encapsulation-dehydration were validated by five members of the EU research infrastructure consortium, COBRA, and two independent external validators. The test strain Chlorella vulgaris SAG 211-11b was successfully cryopreserved using two-step cooling employing passive (Mr Frosty) and Controlled Rate Freezers (CRF) attaining the desired recovery target within 15% of the median viability level (94%). Significant differences (p < 0.05) between cooling regimes were observed where Mr Frosty was more variable (Inter-Quartile Range being 21.5%, versus 13.0% for CRF samples). Viability assessment using fluorescein diacetate gave significantly (P < 0.0001) higher survival than growth in agar with median values being 96% and 89%, respectively. On employing encapsulation-dehydration, greater variability between some validators was observed, with six labs observing recovery in 100% of the beads (84-95% of cells surviving) and one lab observing survival in 80% of the treated beads. Bead disruption followed by algal growth in agar was considered the most reliable and accurate method of assessing cell survival for encapsulation-dehydration.