Comprehensive understanding of the mutant 'giant' Arthrospira platensis developed via ultraviolet mutagenesis.

Introduction: Cyanobacteria are typically of a size that can be observed under a microscope. Here, we present cyanobacteria of a size that can be observed with the naked eye. Arthrospira platensis NCB002 strain showed differentiated morphological characteristics compared to previously reported Arthrospira spp. Methods: Arthrospira platensis NCB002 was obtained by the UV irradiation of Arthrospira sp. NCB001, which was isolated from freshwater and owned by NCell Co., Ltd. A. platensis NIES-39 was obtained from the National Institute for Environmental Studies (Tsukuba, Japan). We used various analytical techniques to determine its overall characteristics. Results and discussion: The draft genome of strain NCB002 consists of five contigs comprising 6,864,973 bp with a G+C content of 44.3 mol%. The strain NCB002 had an average length of 11.69 ± 1.35 mm and a maximum of 15.15 mm, which is 23.4-50.5 times longer than the length (0.3-0.5 mm) of previously known Arthrospira spp., allowing it to be harvested using a thin sieve. Transcriptome analysis revealed that these morphological differences resulted from changes in cell wall formation mechanisms and increased cell division. Our results show that NCB002 has outstanding industrial value and provides a comprehensive understanding of it.

Genomic Analysis and Assessment of Melanin Synthesis in Amorphotheca resinae KUC3009.

This study reports the draft genome of Amorphotheca resinae KUC30009, a fungal isolate with promising industrial-scale melanin production potential. The mechanisms for melanin or melanin-related pigment formation of this strain were examined through bioinformatic and biochemical strategies. The 30.11 Mb genome of A. resinae contains 9638 predicted genes. Genomic-based discovery analyses identified 14 biosynthetic gene clusters (BGCs) associated with secondary metabolite production. Moreover, genes encoding a specific type 1 polyketide synthase and 4-hydroxynaphthalene reductase were identified and predicted to produce intermediate metabolites of dihydroxy naphthalene (DHN)-melanin biosynthesis pathway, but not to DHN-melanin. These findings were further supported by the detection of increased flaviolin concentrations in mycelia and almost unchanged morphologies of the culture grown with tricyclazole. Apart from this, the formation of melanin in the culture filtrate appeared to depend on the laccase-like activity of multi-copper oxidases. Simultaneously, concentrations of nitrogen-containing sources decreased when the melanin formed in the media. Interestingly, melanin formation in the culture fluid was proportional to laccase-like activity. Based on these findings, we proposed novel strategies for the enhancement of melanin production in culture filtrates. Therefore, our study established a theoretical and methodological basis for synthesizing pigments from fungal isolates using genomic- and biochemical-based approaches.

Application of electrical treatment on Euglena gracilis for increasing paramylon production.

Paramylon also called ß-1,3-glucan is a value-added product produced from Euglena gracilis. Recently, researchers have developed various strategies for the enhanced paramylon production, among which electrical treatment for microbial stimulation can be an alternative owing to the applicability to large-scale cultivation. In this study, we applied the electrical treatment for enhanced paramylon production and found the proper treatment conditions. Under the treatment with platinum electrodes at 10 mA, the paramylon production of treated cells was significantly increased about 2.5-fold, compared to those of the untreated cells, although the density of cells was maintained due to considerable stress. The size of treated cells became larger, possibly due to the increased level of paramylon production within the cells. Accordingly, the contents of glucose uptake, glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), and uridine diphosphoglucose (UDPG) were shifted to appropriate states for the process of paramylon synthesis under the treatment. The increased level of transcripts encoding glucan synthase-like 2 (EgGSL2) was also confirmed via droplet digital PCR (ddPCR) under the treatment. Overall, this study makes a major contribution to research on electrical stimulation and provides new insights into E. gracilis metabolism like paramylon synthesis. KEY POINTS: • Electrical treatment induced the paramylon production and morphological change of Euglena gracilis. • The glucose uptake of E. gracilis was increased during the electrical treatment, fueling the paramylon synthesis.

Genomic analysis of facultatively oligotrophic haloarchaea of the genera Halarchaeum, Halorubrum, and Halolamina, isolated from solar salt.

Extremely halophilic archaea (haloarchaea) belonging to the phylum Euryarchaeota have been found in high-salinity environments. In this study, Halarchaeum sp. CBA1220, Halorubrum sp. CBA1229, and Halolamina sp. CBA1230, which are facultatively oligotrophic haloarchaea, were isolated from solar salt by culture under oligotrophic culture conditions. The complete genomes of strains CBA1220, CBA1229, and CBA1230 were sequenced and were found to contain 3,175,875, 3,582,278, and 3,465,332 bp, with a G + C content of 68.25, 67.66, and 66.75 mol %, respectively. In total, 60, 36, and 33 carbohydrate-active enzyme genes were determined in the respective strains. The strains harbored various genes encoding stress-tolerance proteins, including universal stress proteins, cold-shock proteins, and rubrerythrin and rubrerythrin-related proteins. The genome data produced in this study will facilitate further research to improve our understanding of other halophilic strains and promote their industrial application.

Astaxanthin biosynthesis promotion with pH shock in the green microalga, Haematococcus lacustris.

In this study, the use of pH shock to improve astaxanthin synthesis in Haematococcus lacustris was investigated. It has been found that pH shock (pH = 4.5, 60 s) imposes stress in the cells and induces physiological changes, which result in astaxanthin accumulation. The optimal acid-base combination of pH shock was H2SO4-KOH, which increased the astaxanthin content per cell to 39 ± 6.92% than those of the control. In addition, pH shock can be applied simultaneously with the other inductive strategies such as high irradiance and carbon source supply. When high irradiance was applied simultaneously with pH shock, astaxanthin yield was increased 65 ± 0.541% than control. In addition, astaxanthin content per cell was increased 105 ± 6.66% than those of the control, with the concomitant application of carbon source addition with pH shock. Herein, these novel findings provide a useful technique for producing astaxanthin using H. lacustris.

The influence of surrounding land cover on wetland habitat conditions: a case study of inland wetlands in South Korea.

Wetland ecosystems have been globally degraded and lost due to rapid urbanization and climate change. An assessment of national scale inventory, including wetland types and conditions, is urgently required to understand the big picture of endangered wetlands, such as where they are and how they look like. We analyzed the spatial patterns of each inland wetland type (brackish wetland was included) in South Korea and the relative importance of land cover categories on wetland conditions. The wetlands were grouped into four dominant types (riverine, lake, mountain, and human-made) according to their topography. Riverine wetlands constituted the largest area (71.3%). The relative ratio of wetlands in a well-conserved condition (i.e., "A" rank) was highest in riverine wetlands (23.8%), followed by mountain wetlands (22.1%). The higher proportion of grasslands was related to a better condition ranking, but the increasing bareland area had a negative impact on wetland conditions. We also found that wetlands located near wetland protected areas tend to be in a better condition compared to remote sites. Our results further support the importance of the condition of surrounding areas for wetland conservation.

Raineyella fluvialis sp. nov., an actinobacterium isolated from freshwater sediment.

A novel, facultatively anaerobic actinobacterium, designated strain CBA3103T, was isolated from sediment of the Geum River in South Korea. Phylogenetic analysis indicated that strain CBA3103T is most closely related to Raineyella antarctica LZ-22T (98.47 % 16S rRNA gene sequence similarity). The genome of strain CBA3103T was 3 649 865 bp with a DNA G+C content of 69.6 mol%. The average nucleotide identity value between strain CBA3103T and R. antarctica LZ-22T was 79.22 %. Cells of strain CBA3103T were Gram-positive, rod-shaped, 0.6-0.9 µm wide and 1.4-2.4 µm long. Growth occurred at 15-40 °C (optimum, 35 °C), at pH 6.0-7.0 (optimum, pH 7.0) and with 0-2 % NaCl (w/v) (optimum, 0-1 %, w/v). The major cellular fatty acids in strain CBA3103T were anteiso-C15 : 0, anteiso-C15 : 1 A and iso-C14 : 0. The major respiratory quinone was menaquinone-9(H4). The polar lipids of strain CBA3103T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, five unidentified glycolipids and three unidentified phospholipids. Based on the genotypic, phenotypic and chemotaxonomic analyses, strain CBA3103T represents a novel species of the genus Raineyella, for which the name Raineyella fluvialis sp. nov. (type strain CBA3103T=KACC 21446T=DSM 110288T) is proposed.

Characterization of a potential probiotic bacterium Lactococcus raffinolactis WiKim0068 isolated from fermented vegetable using genomic and in vitro analyses.

BACKGROUND: Lactococcus members belonging to lactic acid bacteria are widely used as starter bacteria in the production of fermented dairy products. From kimchi, a Korean food made of fermented vegetables, Lactococcus raffinolactis WiKim0068 was isolated and its genome was analyzed. RESULTS: The complete genome of the strain WiKim0068 consists of one chromosome and two plasmids that comprises 2,292,235 bp, with a G + C content of 39.7 mol%. Analysis of orthoANI values among Lactococcus genome sequences showed that the strain WiKim0068 has > 67% sequence similarity to other species and subspecies. In addition, it displayed no antibiotic resistance and can metabolize nicotinate and nicotinamide (vitamin B3). CONCLUSION: These results augments our understanding of the genus Lactococcus and suggest that this new strain has potential industrial applications.

Control of a toxic cyanobacterial bloom species, Microcystis aeruginosa, using the peptide HPA3NT3-A2.

Microcystis aeruginosa, a species of freshwater cyanobacteria, is known to be one of the dominant species causing cyanobacterial harmful algal blooms (CyanoHABs). M. aeruginosa blooms have the potential to produce neurotoxins and peptide hepatotoxins, such as microcystins and lipopolysaccharides (LPSs). Currently, technologies for CyanoHAB control do not provide any ultimate solution because of the secondary pollution associated with the control measures. In this study, we attempted to use the peptide HPA3NT3-A2, which has been reported to be nontoxic and has antimicrobial properties, for the development of an eco-friendly control against CyanoHABs. HPA3NT3-A2 displayed significant algicidal effects against M. aeruginosa cells. HPA3NT3-A2 induced cell aggregation and flotation (thereby facilitating harvest), inhibited cell growth through sedimentation, and eventually destroyed the cells. HPA3NT3-A2 had no algicidal effect on other microalgal species such as Haematococcus pluvialis and Chlorella vulgaris. Additionally, HPA3NT3-A2 was not toxic to Daphnia magna. The algicidal mechanism of HPA3NT3-A2 was intracellular penetration. The results of this study suggest the novel possibility of controlling CyanoHABs using HPA3NT3-A2.

Community structures and genomic features of undesirable white colony-forming yeasts on fermented vegetables.

White colony-forming yeasts (WCFYs) often appear in fermented foods, depending on the storage method. Despite the ongoing research on fermented foods, the community and genome features of WCFYs have not been well studied. In this study, the community structures of WCFYs on fermented vegetables (kimchi) prepared with various raw materials were investigated using deep sequencing. Only eight operational taxonomic units (OTUs) were detected, indicating that the community structure of WCFYs on kimchi is very simple. The five most abundant OTUs represented Pichia kluyveri, Yarrowia lipolytica, Candida sake, Hanseniaspora uvarum, and Kazachstania servazzii. Using a culture-dependent method, 41 strains representing the five major OTUs were isolated from the surface of the food samples. Whole genomes of the five major yeast strains were sequenced and annotated. The total genome length for the strains ranged from 8.97 Mbp to 21.32 Mbp. This is the first study to report genome sequences of the two yeasts Pichia kluyveri and Candida sake. Genome analysis indicated that each yeast strain had core metabolic pathways such as oxidative phosphorylation; purine metabolism; glycolysis/gluconeogenesis; aminoacyl-tRNA biosynthesis; citrate cycle; but strain specific pathways were also found. In addition, no toxin or antimicrobial resistance genes were identified. Our study provides genome information for five WCFY strains that may highlight their potential beneficial or harmful metabolic effects in fermented vegetables.

Recycling waste nutrient solution originating from the plant factory with the cultivation of newly isolated Acutodesmus species.

Plant factories have been developed to replace traditional agriculture, aiming to solve future problems of food availability. However, the nutrient solution in a plant factory is discharged after a single batch of plant cultivation, giving rise to large amounts of waste nutrient solution. Microalgae can be used to treat a wide variety of wastewater and effectively remove excessive nutrients from wastewater. Therefore, the incorporation of microalgal cultivation into a plant factory to treat waste nutrients would be a reasonable approach facilitating removal of waste nutrients with concomitant production of algal biomass. In this study, we isolated novel microalgal species suitable for the growth in waste nutrients from a plant factory and subsequently an underwater LED photobioreactor was constructed being incorporated into the plant factory system. Finally, treated waste nutrient solution was recycled back into the plant factory and successfully used for the cultivation of plant of butterhead lettuce (Lactuca sativa L.).

Blue-Red LED wavelength shifting strategy for enhancing beta-carotene production from halotolerant microalga, Dunaliella salina.

In the present study, to improve the photosynthetic betacarotene productivity of Dunaliella salina, a blue-red LED wavelength-shifting system (B-R system) was investigated. Dunaliella salina under the B-R system showed enhanced density and beta-carotene productivity compared to D. salina cultivated under single light-emitting diode light wavelengths (blue, white, and red light-emitting diode). Additionally, we developed blue light-adapted D. salina (ALE-D. salina) using an adaptive laboratory evolution (ALE) approach. In combination with the B-R system applied to ALE-D. salina (ALE B-R system), the beta-carotene concentration (33.94 ± 0.52 µM) was enhanced by 19.7% compared to that observed for the non-ALE-treated wild-type of D. salina (intact D. salina) under the B-R system (28.34 ± 0.24 µM).

Salicibibacter kimchii gen. nov., sp. nov., a moderately halophilic and alkalitolerant bacterium in the family Bacillaceae, isolated from kimchi.

A moderately halophilic and alkalitolerant bacterial strain NKC1-1T was isolated from commercial kimchi in Korea. Strain NKC1-1T was Gram-stain-positive, aerobic, rod-shaped, non-motile, and contained diaminopimelic acid-type murein. Cell growth was observed in a medium containing 0-25% (w/v) NaCl (optimal at 10% [w/v]), at 20-40°C (optimal at 37°C) and pH 6.5-10.0 (optimal at pH 9.0). The major isoprenoid quinone of the isolate was menaquinone-7, and the major polar lipids were phosphatidylglycerol and unidentified phospholipids. Cell membrane of the strain contained iso-C17:0 and anteiso-C15:0 as the major fatty acids. Its DNA G + C content was 45.2 mol%. Phylogenetic analysis indicated the strain to be most closely related to Geomicrobium halophilum with 92.7-92.9% 16S rRNA gene sequence similarity. Based on polyphasic taxonomic evaluation with phenotypic, phylogenetic, and chemotaxonomic analyses, the strain represents a novel species in a new genus, for which the name Salicibibacter kimchii gen. nov., sp. nov. is proposed (= CECT 9537T; KCCM 43276T).

Enhancement of microalga Haematococcus pluvialis growth and astaxanthin production by electrical treatment.

In this study, we investigated the effects of electrical treatment on Haematococcus pluvialis growth. The slow growth of H. pluvialis is a major limitation for its mass production. We discovered that electrical treatment may promote the growth of H. pluvialis. To evaluate optimal growth-promoting conditions, the algal growth rate was investigated at various voltages. The optimum current was identified as 100 mA (voltage: 25 V). In comparison with the non-treated cells, those subjected to electrical treatment showed a 1.2 fold increase in cell density. Further experiments confirmed the direct impact of electrical treatment on the growth of H. pluvialis. The periodic application of electrical voltage resulted in a significant increase in the dry weight and astaxanthin production. The astaxanthin content in the periodic application of electrical treatment was 32.6 mg/L, which was a 10% increase compared to those in the non-treated controls. This strategy may serve as a novel approach to enhance H. pluvialis growth as well as astaxanthin production.

Role of jeotgal, a Korean traditional fermented fish sauce, in microbial dynamics and metabolite profiles during kimchi fermentation.

We investigated the effects of jeotgal (fermented fish sauce) on kimchi fermentation, with or without saeu-jeot and myeolchi-jeot. Bacterial community analysis showed that Leuconostoc, Weissella, Lactobacillus, and Tetragenococcus were the dominant genera; however, their succession depended on the presence of jeotgal. Leuconostoc gasicomitatum was the dominant species in kimchi without jeotgal, whereas Weissella koreensis and Lactobacillus sakei were the dominant species in kimchi with myeolchi-jeot and saeu-jeot, respectively. Metabolite analysis, using 1H NMR, showed that the amounts of amino acids and gamma-aminobutyric acid (GABA) were higher in kimchi with jeotgal. Increases in acetate, lactate, and mannitol contents depended on fructose consumption and were more rapid in kimchi with jeotgal. Moreover, the consumption of various amino acids affected the increase in kimchi LAB. Thus, the role of jeotgal in kimchi fermentation was related to enhancement of taste, the amino acid source, and the increases in levels of functional metabolites.

Comparative transcriptome analysis of Haematococcus pluvialis on astaxanthin biosynthesis in response to irradiation with red or blue LED wavelength.

The unicellular green microalga Haematococcus pluvialis has the highest content of the natural antioxidant, astaxanthin. Previously, it was determined that astaxanthin accumulation in H. pluvialis could be induced by blue-wavelength irradiation; however, the molecular mechanism remains unknown. The present study aimed to compare the transcriptome of H. pluvialis, with respect to astaxanthin biosynthesis, under the monochromatic red (660 nm) or blue (450 nm) light-emitting diode (LED) irradiation. Among a total of 165,372 transcripts, we identified 67,703 unigenes, of which 2245 and 171 were identified as differentially expressed genes (DEGs) in response to blue and red irradiation, respectively. Interestingly, expressional changes of blue light receptor cryptochromes were detected in response to blue and/or red LED irradiation in H. pluvialis, which may directly and indirectly regulate astaxanthin biosynthesis. In accordance with this observation, expression of the BKT and CHY genes, which are part of the downstream section of the astaxanthin biosynthetic pathway, was significantly upregulated by blue LED irradiation compared with their expression under control white irradiation. Contrastingly, they were downregulated by red LED irradiation. Our transcriptome study provided molecular insights that highlighted the different of responses of H. pluvialis to red and blue irradiation, especially for astaxanthin biosynthesis.

Novel haloarchaeon Natrinema thermophila having the highest growth temperature among haloarchaea with a large genome size.

Environmental temperature is one of the most important factors for the growth and survival of microorganisms. Here we describe a novel extremely halophilic archaeon (haloarchaea) designated as strain CBA1119T isolated from solar salt. Strain CBA1119T had the highest maximum and optimal growth temperatures (66 °C and 55 °C, respectively) and one of the largest genome sizes among haloarchaea (5.1 Mb). It also had the largest number of strain-specific pan-genome orthologous groups and unique pathways among members of the genus Natrinema in the class Halobacteria. A dendrogram based on the presence/absence of genes and a phylogenetic tree constructed based on OrthoANI values highlighted the particularities of strain CBA1119T as compared to other Natrinema species and other haloarchaea members. The large genome of strain CBA1119T may provide information on genes that confer tolerance to extreme environmental conditions, which may lead to the discovery of other thermophilic strains with potential applications in industrial biotechnology.