Algal Lysis by Sagittula stellata for the Production of Intracellular Valuables.

The purpose of this study was to examine the efficacy of the algicidal bacterium Sagittula stellata on the cell lysis of Nannochloropsis oceanica, a microalga found in the marine environment, in order to extract intracellular valuables. Algicidal bacteria are capable of lysing algal cell walls while keeping lipids and proteins intact yet separated. We obtained these microbes from locations with consistent algae blooms and found that the bacterium Sagittula stellata displayed significant algicidal properties toward Nannochloropsis oceanica, achieving an algicidal rate of 80.1%. We detected a decrease of 66.2% in in vivo fluorescence intensity in algae cultures, obtained a recoverable crude lipid content of 23.3% and a polyunsaturated fatty acid (PUFA) ratio of 29.0% of bacteria-treated algae, and observed the lysis of the cell membrane and the structure of the nucleus of algae. We also identified the inhibited transcription of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) gene and proliferating cell nuclear antigen (PCNA)-related genes and the upregulated heat shock protein (hsp) gene in algal cells during bacterial exposure. Our results indicate that Sagittula stellata effectively lysed microalgae cells, allowing the recovery of intracellular valuables. The algicidal method of Sagittula stellata on Nannochloropsis oceanica cells was confirmed to be a direct attack (or predation), followed by an indirect attack through the secretion of extracellular algicidal compounds. This study provides an important framework for the broad application of algicidal microorganisms in algal cell disruption and the production of intracellular valuables.

Antimicrobial Meroterpenoids and Erythritol Derivatives Isolated from the Marine-Algal-Derived Endophytic Fungus Penicillium chrysogenum XNM-12.

One new meroterpenoid-type alkaloid, oxalicine C (1), and two new erythritol derivatives, penicierythritols A (6) and B (7), together with four known meroterpenoids (2-5), were isolated from the marine algal-derived endophytic fungus Penicillium chrysogenum XNM-12. Their planar structures were determined by means of spectroscopic analyses, including UV, 1D and 2D NMR, and HRESIMS spectra. Their stereochemical configurations were established by comparing the experimental and calculated electronic circular dichroism (ECD) spectra for compound 1, as well as by comparison of the optical rotations with literature data for compounds 6 and 7. Notably, oxalicine C (1) represents the first example of an oxalicine alkaloid with a cleaved α-pyrone ring, whereas penicierythritols A (6) and B (7) are the first reported from the Penicillium species. The antimicrobial activities of compounds 1-7 were evaluated. Compounds 1 and 6 exhibited moderate antibacterial effects against the plant pathogen Ralstonia solanacearum with minimum inhibitory concentration (MIC) values of 8 and 4 µg/mL, respectively. Compound 6 also possesses moderate antifungal properties against the plant pathogen Alternaria alternata with a MIC value of 8 µg/mL.

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.

Purification and biochemical characterization of two keratinases from Bacillus amyloliquefaciens S13 isolated from marine brown alga Zonaria tournefortii with potential keratin-biodegradation and hide-unhairing activities.

The current paper reports the purification and biochemical characterization of two extracellular keratinolytic enzymes, with moderate elastolytic activity, from Bacillus amyloliquefaciens strain S13 newly isolated from the brown alga Zonaria tournefortii. The enzymes were purified to homogeneity by precipitation with (NH4)2SO4-dialysis, followed by size exclusion HPLC column, and submitted to biochemical characterization assays. The findings revealed that the pure enzymes designated KERZT-A and B were monomers with molecular masses of 28 and 47 kDa, respectively. Their identified NH2-terminal amino acid displayed high homologies with those of Bacillus keratinases. While KERZT-A was optimally active at pH 6.5 and 50 °C, KERZT-B showed optimum activity at pH 8 and 60 °C. Both enzymes were completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests their belonging to the serine keratinases family. Interestingly, KERZT-A displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than KERUS from Brevibacillus brevis strain US575, NUE 12 MG (commercial enzyme), and KERZT-B unhairing keratinases. Above all, the findings indicated that KERZT-A and B enzymes seems to be an effective and an eco-friendly alternative to the conventional chemicals used for the feather keratin-biodegradation and for the unhairing of hides or skins in the leather processing industry.

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.

Fungal-bacterial dynamics and their contribution to terrigenous carbon turnover in relation to organic matter quality.

Ecological functions of fungal and bacterial decomposers vary with environmental conditions. However, the response of these decomposers to particulate organic matter (POM) quality, which varies widely in aquatic ecosystems, remains poorly understood. Here we investigated how POM pools of substrates of different qualities determine the relative contributions of aquatic fungi and bacteria to terrigenous carbon (C) turnover. To this end, surface sediments were incubated with different POM pools of algae and/or leaf litter. 13C stable-isotope measurements of C mineralization were combined with phospholipid analysis to link the metabolic activities and substrate preferences of fungal and bacterial heterotrophs to dynamics in their abundance. We found that the presence of labile POM greatly affected the dominance of bacteria over fungi within the degrader communities and stimulated the decomposition of beech litter primarily through an increase in metabolic activity. Our data indicated that fungi primarily contribute to terrigenous C turnover by providing litter C for the microbial loop, whereas bacteria determine whether the supplied C substrate is assimilated into biomass or recycled back into the atmosphere in relation to phosphate availability. Thus, this study provides a better understanding of the role of fungi and bacteria in terrestrial-aquatic C cycling in relation to environmental conditions.

Longitudinal Analysis of Microbiota in Microalga Nannochloropsis salina Cultures.

Large-scale open microalgae cultivation has tremendous potential to make a significant contribution to replacing petroleum-based fuels with biofuels. Open algal cultures are unavoidably inhabited with a diversity of microbes that live on, influence, and shape the fate of these ecosystems. However, there is little understanding of the resilience and stability of the microbial communities in engineered semicontinuous algal systems. To evaluate the dynamics and resilience of the microbial communities in microalgae biofuel cultures, we conducted a longitudinal study on open systems to compare the temporal profiles of the microbiota from two multigenerational algal cohorts, which include one seeded with the microbiota from an in-house culture and the other exogenously seeded with a natural-occurring consortia of bacterial species harvested from the Pacific Ocean. From these month-long, semicontinuous open microalga Nannochloropsis salina cultures, we sequenced a time-series of 46 samples, yielding 8804 operational taxonomic units derived from 9,160,076 high-quality partial 16S rRNA sequences. We provide quantitative evidence that clearly illustrates the development of microbial community is associated with microbiota ancestry. In addition, N. salina growth phases were linked with distinct changes in microbial phylotypes. Alteromonadeles dominated the community in the N. salina exponential phase whereas Alphaproteobacteria and Flavobacteriia were more prevalent in the stationary phase. We also demonstrate that the N. salina-associated microbial community in open cultures is diverse, resilient, and dynamic in response to environmental perturbations. This knowledge has general implications for developing and testing design principles of cultivated algal systems.

Selective growth promotion of bloom-forming raphidophyte Heterosigma akashiwo by a marine bacterial strain.

Algal bloom is typically caused by aberrant propagation of a single species, resulting in its predomination in the local population. While environmental factors including temperature and eutrophication are linked to bloom, the precise mechanism of its formation process is still obscure. Here, we isolated a bacterial strain that promotes growth of Heterosigma akashiwo, a Raphidophyceae that causes harmful algal blooms. Based on 16S rRNA gene sequence, the strain was identified as Altererythrobacter ishigakiensis, a member of the class Alphaproteobacteria. When added to culture, this strain facilitated growth of H. akashiwo and increased its cell culture yield significantly. Importantly, this strain did not affect the growth of other raphidophytes, Chattonella ovate and C. antiqua, indicating that it promotes growth of H. akashiwo in a species-specific manner. We also found that, in co-culture, H. akashiwo suppressed the growth of C. ovate. When A. ishigakiensis was added to the mixed culture, H. akashiwo growth was facilitated while C. ovate propagation was markedly suppressed, indicating that the presence of the bacterium enhances the dominance of H. akashiwo over C. ovate. This is the first example of selective growth promotion of H. akashiwo by a marine bacterium, and may exemplify importance of symbiotic bacterium on algal bloom forming process in general.

Community-Level and Species-Specific Associations between Phytoplankton and Particle-Associated Vibrio Species in Delaware's Inland Bays.

Vibrio species are an abundant and diverse group of bacteria that form associations with phytoplankton. Correlations between Vibrio and phytoplankton abundance have been noted, suggesting that growth is enhanced during algal blooms or that association with phytoplankton provides a refuge from predation. Here, we investigated relationships between particle-associated Vibrio spp. and phytoplankton in Delaware's inland bays (DIB). The relative abundances of particle-associated Vibrio spp. and algal classes that form blooms in DIB (dinoflagellates, diatoms, and raphidophytes) were determined using quantitative PCR. The results demonstrated a significant correlation between particle-associated Vibrio abundance and phytoplankton, with higher correlations to diatoms and raphidophytes than to dinoflagellates. Species-specific associations were examined during a mixed bloom of Heterosigma akashiwo and Fibrocapsa japonica (Raphidophyceae) and indicated a significant positive correlation for particle-associated Vibrio abundance with H. akashiwo but a negative correlation with F. japonica. Changes in Vibrio assemblages during the bloom were evaluated using automated ribosomal intergenic spacer analysis (ARISA), which revealed significant differences between each size fraction but no significant change in Vibrio assemblages over the course of the bloom. Microzooplankton grazing experiments showed that losses of particle-associated Vibrio spp. may be offset by increased growth in the Vibrio population. Moreover, analysis of Vibrio assemblages by ARISA also indicated an increase in the relative abundance for specific members of the Vibrio community despite higher grazing pressure on the particle-associated population as a whole. The results of this investigation demonstrate links between phytoplankton and Vibrio that may lead to predictions of potential health risks and inform future management practices in this region.

Three new asperentin derivatives from the algicolous fungus Aspergillus sp. F00785.

Three new asperentin-type compounds, 6-O-α-d-ribosylasperentin (1) and 6-O-α-d-ribosyl-8-O-methylasperentin (2) and 5-hydroxyl-6-O-methylasperentin (3), along with asperentin (4) and its known analogues (5-9), were isolated from a halotolerant Aspergillus sp. strain F00785, an endotrophic fungus from marine alga. Their structures were determined using extensive NMR and HRESIMS spectroscopic analysis, including the X-ray crystallographic data for the assignment of the absolute configurations of compound 9. Compound 4 exhibited highly potent inhibitory activity against crop pathogens, Colletotrichum gleosporioides Penz. and Colletotrichum gleosporioides (Penz.) Sacc.

16S rDNA-based metagenomic analysis of bacterial diversity associated with two populations of the kleptoplastic sea slug Elysia chlorotica and its algal prey Vaucheria litorea.

The molluscan sea slug Elysia chlorotica is best known for its obligate endosymbiosis with chloroplasts (= kleptoplasty) from its algal prey Vaucheria litorea and its ability to sustain itself photoautotrophically for several months. This unusual photosynthetic sea slug also harbors an array of undescribed bacteria, which may contribute to the long-term success of the symbiosis. Here, we utilized 16S rDNA-based metagenomic analyses to characterize the microbial diversity associated with two populations of E. chlorotica from Halifax, Nova Scotia, Canada, and from Martha's Vineyard, Massachusetts, USA. Animals were examined immediately after collection from their native environments, after being starved of their algal prey for several months, and after being bred in the laboratory (second-generation sea slugs) to characterize the effect of varying environmental and culturing conditions on the associated bacteria. Additionally, the microbiome of the algal prey, laboratory-cultured V. litorea, was analyzed to determine whether the laboratory-bred sea slugs obtained bacteria from their algal food source during development. Bacterial profiles varied between populations and among all conditions except for the F2 laboratory-bred samples, which were similar in diversity and abundance, but not to the algal microbiome. Alpha-, beta-, and gamma-proteobacteria dominated all of the samples along with Actinobacteria, Bacilli, Flavobacteria, and Sphingobacteria. Bacteria capable of polysaccharide digestion and photosynthesis, as well as putative nitrogen fixation, vitamin B(12) production, and natural product biosynthesis were associated with the sea slug and algal samples.

Phaeobacter gallaeciensis reduces Vibrio anguillarum in cultures of microalgae and rotifers, and prevents vibriosis in cod larvae.

Phaeobacter gallaeciensis can antagonize fish-pathogenic bacteria in vitro, and the purpose of this study was to evaluate the organism as a probiont for marine fish larvae and their feed cultures. An in vivo mechanism of action of the antagonistic probiotic bacterium is suggested using a non-antagonistic mutant. P. gallaeciensis was readily established in axenic cultures of the two microalgae Tetraselmis suecica and Nannochloropsis oculata, and of the rotifer Brachionus plicatilis. P. gallaeciensis reached densities of 10(7) cfu/ml and did not adversely affect growth of algae or rotifers. Vibrio anguillarum was significantly reduced by wild-type P. gallaeciensis, when introduced into these cultures. A P. gallaeciensis mutant that did not produce the antibacterial compound tropodithietic acid (TDA) did not reduce V. anguillarum numbers, suggesting that production of the antibacterial compound is important for the antagonistic properties of P. gallaeciensis. The ability of P. gallaeciensis to protect fish larvae from vibriosis was determined in a bath challenge experiment using a multidish system with 1 larva per well. Unchallenged larvae reached 40% accumulated mortality which increased to 100% when infected with V. anguillarum. P. gallaeciensis reduced the mortality of challenged cod larvae (Gadus morhua) to 10%, significantly below the levels of both the challenged and the unchallenged larvae. The TDA mutant reduced mortality of the cod larvae in some of the replicates, although to a much lesser extent than the wild type. It is concluded that P. gallaeciensis is a promising probiont in marine larviculture and that TDA production likely contributes to its probiotic effect.

Mixotrophy in red tide algae raphidophytes.

Marine raphidophytes are common red tide organisms that are distributed worldwide. They are known to be harmful to other plankton and fish and have often caused large-scale fish mortality in many countries. Thus, the population dynamics of raphidophytes is a critical concern for scientists, the aquaculture industry, and government officers from many countries. Raphidophyte growth and mortality should be investigated to understand bloom dynamics. Raphidophytes were thought to be exclusively autotrophic organisms. However, several recent studies have revealed that raphidophytes are able to feed on heterotrophic and autotrophic bacteria, i.e. raphidophytes are mixotrophic algae. Further, high-resolution video microscopy has revealed the mechanism by which raphidophytes feed on bacteria, which involves capturing prey cells in the mucus excreted by mucocysts and engulfing the cells through mucocysts. These discoveries may influence the conventional view on both raphidophyte bloom dynamics and plankton energy flow and carbon cycling. In the present study, I review prey, feeding mechanisms, and ingestion rates of mixotrophic marine raphidophytes. In addition, I examine the ecological significance of raphidophyte mixotrophy.

Use of an antifungal drug, amphotericin B for isolation of thraustochytrids.

The inhibitory effect of amphotericin B (AMPH) on the growth of fungi during the isolation of thraustochytrids was examined. The growth of fungi was significantly inhibited by addition of AMPH, and therefore colonies of thraustochytrids were not overlaid with fungal mycelia, which resulted in increased efficiency of thraustochytrids isolation.