Synthesis of Silver Nanoparticles Using Aggregatimonas sangjinii F202Z8T and Their Biological Characterization.

The aim of this study is to describe the general features and eco-friendly biosynthesis of silver nanoparticles (AgNPs) from the marine bacterium Aggregatimonas sangjinii F202Z8T. To the best of our knowledge, no previous study has reported the biosynthesis of AgNPs using this strain. The formation of AgNPs using F202Z8T was synthesized intracellularly without the addition of any disturbing factors, such as antibiotics, nutrient stress, or electron donors. The AgNPs were examined using UV-vis spectrophotometry, transmission electron microscopy, energy-dispersive X-ray spectroscopy, nanoparticle tracking analysis, and Fourier transform infrared spectrometry. The UV-vis spectrum showed a peak for the synthesized AgNPs at 465 nm. The AgNPs were spherical, with sizes ranging from 27 to 82 nm, as denoted by TEM and NTA. FTIR showed various biomolecules including proteins and enzymes that may be involved in the synthesis and stabilization of AgNPs. Notably, the AgNPs demonstrated broad-spectrum antibacterial effects against various pathogenic Gram-positive and Gram-negative bacteria, including Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. The minimum inhibitory concentrations and minimum bactericidal concentrations of the F202Z8T-formed AgNPs were 80 and 100 µg/mL, 40 and 50 µg/mL, and 30 and 40 µg/mL against E. coli, B. subtilis, and S. aureus, respectively. This study suggests that A. sangjinii F202Z8T is a candidate for the efficient synthesis of AgNPs and may be suitable for the formulation of new types of bactericidal substances.

Falsirhodobacter algicola sp. nov., a member of the Rhodobacteraceae isolated from the marine red algae Grateloupia sp.

A Gram-negative, pale yellow-pigmented, non-flagellated, motile, rod-shaped and aerobic bacterium, designated strain PG104T, was isolated from red algae Grateloupia sp. collected from the coastal area of Pohang, Republic of Korea. Growth of strain PG104T was observed at 15-35 °C (optimum, 30 °C), pH 6.0-10.0 (optimum, pH 7.5-8.0) and in the presence of 0-8.0 % (w/v) NaCl (optimum, 5.0 %). The predominant fatty acids included C17 : 0, C18 : 0, 11-methyl C18 : 1 ω7c and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and the major respiratory quinone was Q-10. Polar lipids included phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified lipid and one unidentified aminolipid. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain PG104T formed a phylogenetic lineage with members of the genus Falsirhodobacter and exhibited 16S rRNA gene sequence similarities of 97.1 and 96.6 % to Falsirhodobacter deserti W402T and Falsirhodobacter halotolerans JA744T, respectively. The complete genome of strain PG104T consisted of a single circular chromosome of approximately 2.8 Mbp with five plasmids. Based on polyphasic taxonomic data, strain PG104T represents a novel species in the genus Falsirhodobacter, for which the name Falsirhodobacter algicola sp. nov. is proposed. The type strain of Falsirhodobacter algicola is PG104T (=KCTC 82230T=JCM 34380T).

Aggregatimonas sangjinii gen. nov., sp. nov., a novel silver nanoparticle synthesizing bacterium belonging to the family Flavobacteriaceae.

Microbially synthesized nanoparticles has received increasing attentions owing to the broad applications in biology and medicine. In this study, we report a novel bacterium that biologically generates silver nanoparticles (AgNPs). This bacterium, designated strain F202Z8T, was isolated from a rusty iron plate found in the intertidal region of Taean, South Korea. The morphological, biochemical and molecular characteristics predicted that strain F202Z8T belongs to the family Flavobacteriaceae. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain F202Z8T forms a distinct lineage with closely related genera Maribacter, Pelagihabitans, Pseudozobellia, Zobellia, Pricia, and Costertonia and showed the highest similarity to Maribacter aestuarii GY20T (94.5%). The digital DNA-DNA hybridization and average nucleotide identity values calculated from the whole genome-sequence comparison between strain F202Z8T and other members of the family Flavobacteriaceae were in the ranges of 12.7%-16.9% and 70.3%-74.4%, respectively, suggesting that strain F202Z8T represented a novel genus. The complete genome sequence of strain F202Z8T is 4,723,614 bp, with 43.26% G + C content. Based on the COG, GO, KEGG, NR, and Swiss-Prot databases, the genomic analysis of F202Z8T revealed the presence of 17 putative genes responsible for the synthesis of AgNPs. Our polyphasic taxonomic results suggested that this strain represents a novel species of a novel genus in the family Flavobacteriaceae, for which the name Aggregatimonas sangjinii gen. nov., sp. nov. is proposed. The type strain of Aggregatimonas sangjinii is F202Z8T (= KCCM 43411T = LMG 31494T). Overall, our data provide fundamental information to potentially utilize this novel bacterium for synthesis of nanoparticles.

Exploring the Potential of Nannochloropsis sp. Extract for Cosmeceutical Applications.

Recently, there has been emerging interest in various natural products with skin protective effects as they are recognized as safe and efficient. Microalgae have developed chemical defense systems to protect themselves against oxidative stress caused by UV radiation by producing various bioactive compounds including a number of secondary metabolites, which have potential for cosmeceutical applications. In addition, microalgae have various advantages as a sustainable source for bioactive compounds with diverse functions due to their rapid growth rate, high productivity, and use of non-arable land. In this study, we aimed to investigate the cosmeceutical potential of ethanol extract from Nannochloropsis sp. G1-5 (NG15) isolated from the southern West Sea of the Republic of Korea. It contained PUFAs (including EPA), carotenoids (astaxanthin, canthaxanthin, ß-carotene, zeaxanthin, violaxanthin), and phenolic compounds, which are known to have various skin protective functions. We confirmed that the NG15 extract showed various skin protective functions with low cytotoxicity, specifically anti-melanogenic, antioxidant, skin-moisturizing, anti-inflammatory, anti-wrinkling, and UV protective function, by measuring tyrosinase inhibition activity; melanin content; DPPH radical scavenging activity; expression of HAS-2, MMP-1, and Col1A1 genes; and elastase inhibition activity as well as cell viability after UV exposure. Our results indicated that the NG15 extract has the potential to be used for the development of natural cosmetics with a broad range of skin protective functions.

mCherry Protein as an In Vivo Quantitative Reporter of Gene Expression in the Chloroplast of Chlamydomonas reinhardtii.

Microalgal chloroplasts have a substantial potential as a sustainable alternative to conventional hosts for recombinant protein production, due to their photosynthetic ability. However, realization of microalgal chloroplast as a platform for the production of recombinant proteins has suffered from difficulties in genetic manipulation and development of molecular tools, including reporter proteins. Here, we investigated the suitability of a fluorescent protein, mCherry, as a reporter for quantitative in vivo monitoring of gene expression in the chloroplast of Chlamydomonas reinhardtii. By analyzing cell growth, the fluorescence intensity of a mCherry-expressing strain, as well as auto-fluorescence, under different photoautotrophic culture conditions, we demonstrated a strong correlation between the fluorescence intensity of mCherry expressed in the chloroplast and its protein expression level. In addition, we found that the supply of CO2 and light energy can be an important factor for the synthesis of recombinant proteins in the microalgal chloroplast. Our results identified mCherry as a reliable and quantitative reporter for the study of gene expression in chloroplasts, which is essential for the biotechnological application of microalgal chloroplasts and for improved production of valuable recombinant proteins.

Targeted knockout of phospholipase A2 to increase lipid productivity in Chlamydomonas reinhardtii for biodiesel production.

Biofuel derived from microalgae have several advantages over other oleaginous crops, however, still needs to be improved with its cost aspect and can be achieved by developing of a strain with improved lipid productivity. In this study, the CRISPR-Cas9 system was incorporated to carry out a target-specific knockout of the phospholipase A2 gene in Chlamydomonas reinhardtii. The targeted gene encodes a key enzyme in the Lands cycle. As a result, the mutants showed a characteristic of increased diacylglycerol pool, followed by a higher accumulation of triacylglycerol without being significantly compensated with the cell growth. As a result, the overall lipid productivities of phospholipase A2 knockout mutants have increased by up to 64.25% (to 80.92 g L-1 d-1). This study can provide crucial information for the biodiesel industry.

Manipulation of the microalgal chloroplast by genetic engineering for biotechnological utilization as a green biofactory.

The chloroplast is an essential organelle in microalgae for conducting photosynthesis, thus enabling the photoautotrophic growth of microalgae. In addition to photosynthesis, the chloroplast is capable of various biochemical processes for the synthesis of proteins, lipids, carbohydrates, and terpenoids. Due to these attractive characteristics, there has been increasing interest in the biotechnological utilization of microalgal chloroplast as a sustainable alternative to the conventional production platforms used in industrial biotechnology. Since the first demonstration of microalgal chloroplast transformation, significant development has occurred over recent decades in the manipulation of microalgal chloroplasts through genetic engineering. In the present review, we describe the advantages of the microalgal chloroplast as a production platform for various bioproducts, including recombinant proteins and high-value metabolites, features of chloroplast genetic systems, and the development of transformation methods, which represent important factors for gene expression in the chloroplast. Furthermore, we address the expression of various recombinant proteins in the microalgal chloroplast through genetic engineering, including reporters, biopharmaceutical proteins, and industrial enzymes. Finally, we present many efforts and achievements in the production of high-value metabolites in the microalgal chloroplast through metabolic engineering. Based on these efforts and advances, the microalgal chloroplast represents an economically viable and sustainable platform for biotechnological applications in the near future.

Euzebyella algicola sp. nov., a marine bacterium of the family Flavobacteriaceae, isolated from green algae.

A Gram-negative, yellow-pigmented, non-flagellated, gliding, rod-shaped and aerobic bacterium, designated MEBiC 12267T, was isolated from green algae of Jeju Island. 16S rRNA gene sequence analysis revealed that the strain MEBiC 12267T was affiliated to the genus Euzebyella of the family Flavobacteriaceae and showed the highest similarity to Euzebyella marina KCTC 42440T (98.5 %). The DNA-DNA relatedness value of strain MEBiC 12267T with E. marina KCTC 42440T was 25 %. Growth was observed at 10-37 °C (optimum, 30-33 °C), at pH 6.0-9.5 (optimum, 8.0-8.5) and with 0.5-9.0 % (w/v) NaCl (optimum, 2.5-3.5 %). The predominant cellular fatty acids were iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The major respiratory quinone was MK-6. Polar lipids included phosphatidylethanolamine, seven unidentified lipids and two unidentified aminolipids. The DNA G+C content was 40.7 mol%. On the basis of the data from the polyphasic taxonomic study, it was concluded that the strain MEBiC 12267T represents a novel species within the genus Euzebyella, for which the name Euzebyella algicola sp. nov. is proposed. The type strain of E. algicola is MEBiC 12267T (=KCCM 43264T=JCM 32170T).

Magnetophoretic sorting of microdroplets with different microalgal cell densities for rapid isolation of fast growing strains.

Microalgae - unicellular photosynthetic organisms - have received increasing attention for their ability to biologically convert CO2 into valuable products. The commercial use of microalgae requires screening strains to improve the biomass productivity to achieve a high-throughput. Here, we developed a microfluidic method that uses a magnetic field to separate the microdroplets containing different concentrations of microalgal cells. The separation efficiency is maximized using the following parameters that influence the amount of lateral displacement of the microdroplets: magnetic nanoparticle concentration, flow rate of droplets, x- and y-axis location of the magnet, and diameter of the droplets. Consequently, 91.90% of empty, 87.12% of low-, and 90.66% of high-density droplets could be separated into different outlets through simple manipulation of the magnetic field in the microfluidic device. These results indicate that cell density-based separation of microdroplets using a magnetic force can provide a promising platform to isolate microalgal species with a high growth performance.

Quantitative analysis of the chemotaxis of a green alga, Chlamydomonas reinhardtii, to bicarbonate using diffusion-based microfluidic device.

There is a growing interest in the photosynthetic carbon fixation by microalgae for the production of valuable products from carbon dioxide (CO2). Microalgae are capable of transporting bicarbonate (HCO3 (-)), the most abundant form of inorganic carbon species in the water, as a source of CO2 for photosynthesis. Despite the importance of HCO3 (-) as the carbon source, little is known about the chemotactic response of microalgae to HCO3 (-). Here, we showed the chemotaxis of a model alga, Chlamydomonas reinhardtii, towards HCO3 (-) using an agarose gel-based microfluidic device with a flow-free and stable chemical gradient during the entire assay period. The device was validated by analyzing the chemotactic responses of C. reinhardtii to the previously known chemoattractants (NH4Cl and CoCl2) and chemotactically neutral molecule (NaCl). We found that C. reinhardtii exhibited the strongest chemotactic response to bicarbonate at the concentration of 26 mM in a microfluidic device. The chemotactic response to bicarbonate showed a circadian rhythm with a peak during the dark period and a valley during the light period. We also observed the changes in the chemotaxis to bicarbonate by an inhibitor of bicarbonate transporters and a mutation in CIA5, a transcriptional regulator of carbon concentrating mechanism, indicating the relationship between chemotaxis to bicarbonate and inorganic carbon metabolism in C. reinhardtii. To the best of our knowledge, this is the first report of the chemotaxis of C. reinhardtii towards HCO3 (-), which contributes to the understanding of the physiological role of the chemotaxis to bicarbonate and its relevance to inorganic carbon utilization.

Microfluidic high-throughput selection of microalgal strains with superior photosynthetic productivity using competitive phototaxis.

Microalgae possess great potential as a source of sustainable energy, but the intrinsic inefficiency of photosynthesis is a major challenge to realize this potential. Photosynthetic organisms evolved phototaxis to find optimal light condition for photosynthesis. Here we report a microfluidic screening using competitive phototaxis of the model alga, Chlamydomonas reinhardtii, for rapid isolation of strains with improved photosynthetic efficiencies. We demonstrated strong relationship between phototaxis and photosynthetic efficiency by quantitative analysis of phototactic response at the single-cell level using a microfluidic system. Based on this positive relationship, we enriched the strains with improved photosynthetic efficiency by isolating cells showing fast phototactic responses from a mixture of 10,000 mutants, thereby greatly improving selection efficiency over 8 fold. Among 147 strains isolated after screening, 94.6% showed improved photoautotrophic growth over the parental strain. Two mutants showed much improved performances with up to 1.9- and 8.1-fold increases in photoautotrophic cell growth and lipid production, respectively, a substantial improvement over previous approaches. We identified candidate genes that might be responsible for fast phototactic response and improved photosynthesis, which can be useful target for further strain engineering. Our approach provides a powerful screening tool for rapid improvement of microalgal strains to enhance photosynthetic productivity.

Multiplex microfluidic system integrating sequential operations of microalgal lipid production.

The unit cost for the production of algal biofuel needs to be reduced in order to be a substitute for fossil fuel. To achieve this goal, the development of a novel system is needed for a rapid screening of numerous microalgal species to isolate superior strains with the highest lipid productivity. Here, we developed a PDMS-based multiplex microfluidic system with eight chambers and micropillar arrays to expedite multiple steps for lipid sample preparation from different microalgal strains. We could rapidly and efficiently perform sequential operations from cell culture to lipid extraction of eight different microalgal strains simultaneously on a single device without harvesting and purification steps, which are labor- and energy-intensive, by the simple injection of medium and solvent into the central inlet due to the integrated micropillar arrays connecting the chambers and central inlet. The lipid extraction efficiency using this system was comparable (94.5-102.6%) to the conventional Bligh-Dyer method. We investigated the cell growth and lipid productivity of different strains using the microfluidic device. We observed that each strain has a different lipid accumulation pattern according to stress conditions. These results demonstrate that our multiplex microfluidic approach can provide an efficient analytical tool for the rapid analysis of strain performances (e.g. cell growth and lipid productivities) and the determination of the optimal lipid induction condition for each strain.

Microdroplet photobioreactor for the photoautotrophic culture of microalgal cells.

Microalgae, unicellular photoautotrophic microorganisms, have attracted great attention for the production of biofuel and high-value products, but the commercial use of microalgae has been limited by low photosynthetic productivity. To overcome this limitation, it is required to develop an efficient platform for the rapid evaluation of photoautotrophic growth performance and productivity of microalgal strains. Here we describe a droplet-based photobioreactor for high-throughput analysis of the photoautotrophic growth of microalgal cells. By integrating micropillar arrays and adjusting the height of the microchamber, we could accurately monitor the growth kinetics of microalgae in an immobilized microdroplet and improve the transfer rate of CO2 into the microdroplet photobioreactor with an increased contact area between the microdroplet and PDMS surface. The improvement of CO2 transfer into the microdroplet was also confirmed by improved microalgal cell growth and a decrease in pH measured using colorimetric and fluorescence-based assays. The photoautotrophic growth kinetics of Chlorella vulgaris were measured under different CO2 concentrations (ambient, 1%, 2.5%, 5% and 7.5%) and light intensity (35, 55, 100, 150, and 200 µmol photons per m(2) per s) conditions, which are key factors for photoautotrophic growth. Chlorella vulgaris in a microdroplet showed better cell growth performance compared to a flask culture due to the reduced shading effects and improved mass transfer. Finally, we could evaluate the photoautotrophic growth performance of four microalgal strains (Chlorella vulgaris, Chlorella protothecoides, Chlorella sorokiniana and Neochloris oleoabundans) for 120 hours. These results demonstrate that our microdroplet system can be used as an efficient photobioreactor for the rapid evaluation of the photoautotrophic growth of microalgal strains under various conditions.

A Microreactor System for Cultivation of Haematococcus pluvialis and Astaxanthin Production.

Development of efficient culture and monitoring system for cell growth and production of useful materials is required for practical utilization of microalgae. In the present study, we developed a PDMS-based microreactor system for efficient, rapid culture of microalgae and monitoring of cell growth, carotenoid content under diverse culture conditions. Due to advantages of PDMS, we optimized culture conditions (light intensity, pH, nitrate depletion, carbon dioxide concentration) for improving growth rate and astaxanthin productivity in considerably less time compared to conventional culture methods using flask or well plate. In addition, we found that there was a strong linear correlation between fluorescence intensity of astaxanthin stained by Nile red and the astaxanthin content, which can be utilized as a high-throughput screening tool in microfluidic systems. In this study, the growth rate of vegetative Haematococcus pluvialis was improved by 60% in microfluidic chamber than in flask and astaxanthin was produced up to 362 mg/L under the optimal conditions (300 µmol photon/m2/s of light, 7% CO2 (v/v), and pH 7.0) using designed microfluidic devices. This result shows that microfluidic system can provide effective means to address development of microalgal strains including H. pluvialis and bioprocess.

A microscale approach for simple and rapid monitoring of cell growth and lipid accumulation in Neochloris oleoabundans.

Due to the increasing environmental problems caused by the use of fossil fuels, microalgae have been spotlighted as renewable resources to produce biomass and biofuels. Therefore, the investigation of the optimum culture conditions of microalgae in a short time is one of the important factors for improving growth and lipid productivity. Herein, we developed a PDMS-based high-throughput screening system to rapidly and easily determine the optimum conditions for high-density culture and lipid accumulation of Neochloris oleoabundans. Using the microreactor, we were able to find the optimal culture conditions of N. oleoabundans within 5 days by rapid and parallel monitoring growth and lipid induction under diverse conditions of light intensity, pH, CO2 and nitrate concentration. We found that the maximum growth rate (µ max = 2.13 day(-1)) achieved in the microreactor was 1.58-fold higher than that in a flask (µ max = 1.34 day(-1)) at the light intensity of 40 µmol photons m(-2) s(-1), 5 % CO2 (v/v), pH 7.5 and 7 mM nitrate. In addition, we observed that the accumulation of lipid in the microreactor was 1.5-fold faster than in a flask under optimum culture condition. These results show that the microscale approach has the great potential for improving growth and lipid productivity by high-throughput screening of diverse optimum conditions.