Comparison of Metabarcoding and Microscopy Methodologies to Analyze Diatom Communities in Five Estuaries Along the Southern Coast of the Korean Peninsula.

The study of microalgal communities is critical for understanding aquatic ecosystems. These communities primarily comprise diatoms (Heterokontophyta), with two methods commonly used to study them: Microscopy and metabarcoding. However, these two methods often deliver different results; thus, their suitability for analyzing diatom communities is frequently debated and evaluated. This study used these two methods to analyze the diatom communities in identical water samples and compare the results. The taxonomy of the species constituting the diatom communities was confirmed, and both methods showed that species belonging to the orders Bacillariales and Naviculales (class Bacillariophyceae) are the most diverse. In the lower taxonomic levels (family, genus, and species), microscopy tended to show a bias toward detecting diatom species (Nitzschia frustulum, Nitzschia inconspicua, Nitzschia intermedia, Navicula gregaria, Navicula perminuta, Navicula recens, Navicula sp.) belonging to the Bacillariaceae and Naviculaceae families. The results of the two methods differed in identifying diatom species in the communities and analyzing their structural characteristics. These results are consistent with the fact that diatoms belonging to the genera Nitzschia and Navicula are abundant in the communities; furthermore, only the Illumina MiSeq data showed the abundance of the Melosira and Entomoneis genera. The results obtained from microscopy were superior to those of Illumina MiSeq regarding species-level identification. Based on the results obtained via microscopy and Illumina MiSeq, it was revealed that neither method is perfect and that each has clear strengths and weaknesses. Therefore, to analyze diatom communities effectively and accurately, these two methods should be combined.

The Prokaryotic Microalga Limnothrix redekei KNUA012 to Improve Aldehyde Decarbonylase Expression for Use as a Biological Resource.

The prokaryotic microalga Limnothrix redekei KNUA012 isolated from a freshwater bloom sample from Lake Hapcheon, Hapcheon-gun, South Korea, was investigated for its potential as a biofuel feedstock. Microalgae produce straight-chain alkanes/alkenes from acyl carrier protein-linked fatty acyls via aldehyde decarbonylase (AD; EC 1.2.1.3), which can convert aldehyde intermediates into various biofuel precursors, such as alkanes and free fatty acids. In L. redekei KNUA012, long-chain ADs can convert fatty aldehyde intermediates into alkanes. After heterologous AD expression in Escherichia coli (pET28-AD), we identified an AD in L. redekei KNUA012 that can synthesize various alkanes, such as pentadecane (C15H32), 8-heptadecene (C17H34), and heptadecane (C17H36). These alkanes can be directly used as fuels without transesterification. Biodiesel constituents including dodecanoic acid (C13H26O2), tetradecanoic acid (C15H30O2), 9-hexa decenoic acid (C17H32O2), palmitoleic acid (C17H32O2), hexadecanoic acid (C17H34O2), 9-octadecenoic acid (C19H36O2), and octadecanoic acid (C19H38O2) are produced by L. redekei KNUA012 as the major fatty acids. Our findings suggest that Korean domestic L. redekei KNUA012 is a promising resource for microalgae-based biofuels and biofuel feedstock.

The microbial communities (bacteria, algae, zooplankton, and fungi) improved biofloc technology including the nitrogen-related material cycle in Litopenaeus vannamei farms.

Microbes are essential in biofloc technology for controlling nitrogen levels in water. The composition and function of microorganisms with biofloc systems were reported; however, data on microorganisms other than bacteria, such as algae (which are essential in the nitrogen cycle) and zooplankton (which are bacterial and algal predators), remain limited. The microbial communities (including bacteria, algae, zooplankton, and fungi) were investigated in shrimp farms using biofloc technology. Using Illumina MiSeq sequencing, the V4 region of 18S rRNA and the V3-V4 region of 16S rRNA were utilized for the analysis of the eukaryotic and prokaryotic microbial communities. As a result, it was found that the biofloc in the shrimp farm consisted of 48.73%-73.04% eukaryotic organisms and 26.96%-51.27% prokaryotic organisms. In these shrimp farms, prokaryotic microbial communities had higher specie richness and diversity than eukaryotic microbial communities. However, the eukaryotic microbial communities were more abundant than their prokaryotic counterparts, while algae and zooplankton dominated them. It was discovered that the structures of the microbial communities in the shrimp farms seemed to depend on the effects of predation by zooplankton and other related organisms. The results provided the nitrogen cycle in biofloc systems by the algal and bacterial groups in microbial communities.

Effect of Different Cultivation Modes (Photoautotrophic, Mixotrophic, and Heterotrophic) on the Growth of Chlorella sp. and Biocompositions.

In the past, biomass production using microalgae culture was dependent on inorganic carbon sources as microalgae are photosynthetic organisms. However, microalgae utilize both organic and inorganic carbon sources, such as glucose. Glucose is an excellent source of organic carbon that enhances biomass yield and the content of useful substances in microalgae. In this study, photoautotrophic, mixotrophic, and heterotrophic cultivation conditions were applied to three well-known strains of Chlorella (KNUA104, KNUA114, and KNUA122) to assess biomass productivity, and compositional changes (lipid, protein, and pigment) were evaluated in BG11 media under photoautotrophic, mixotrophic, and heterotrophic conditions utilizing different initial concentrations of glucose (5, 10, 15, 20, and 25 g L-1). Compared to the photoautotrophic condition (biomass yield: KNUA104, 0.35 ± 0.04 g/L/d; KNUA114, 0.40 ± 0.08 g/L/d; KNUA122, 0.38 ± 0.05 g/L/d) glucose was absent, and the biomass yield improved in the mixotrophic (glucose: 20 g L-1; biomass yield: KNUA104, 2.99 ± 0.10 g/L/d; KNUA114, 5.18 ± 0.81 g/L/d; KNUA122, 5.07 ± 0.22 g/L/d) and heterotrophic conditions (glucose: 20 g L-1; biomass yield: KNUA104, 1.72 ± 0.26 g/L/d; KNUA114, 4.26 ± 0.27 g/L/d; KNUA122, 4.32 ± 0.32 g/L/d). All strains under mixotrophic and heterotrophic conditions were optimally cultured when 15-20 g L-1 initial glucose was provided. Although bioresourse productivity improved under both mixotrophic and heterotrophic conditions where mixotrophic conditions were found to be optimal as the yields of lipid and pigment were also enhanced. Protein content was less affected by the presence of light or the concentration of glucose. Under mixotrophic conditions, the highest lipid content (glucose: 15 g L-1; lipid content: 68.80 ± 0.54%) was obtained with Chlorella vulgaris KNUA104, and enhanced pigment productivity of Chlorella sorokiniana KNUA114 and KNUA122 (additional pigment yield obtained with 15 g L-1 glucose: KNUA 114, 0.33 ± 0.01 g L-1; KNUA122, 0.21 ± 0.01 g L-1). Also, saturated fatty acid (SFA) content was enhanced in all strains (SFA: KNUA104, 29.76 ± 1.31%; KNUA114, 37.01 ± 0.98%; KNUA122, 33.37 ± 0.17%) under mixotrophic conditions. These results suggest that mixotrophic cultivation of Chlorella vulgaris and Chlorella sorokiniana could improve biomass yield and the raw material quality of biomass.

Enhancement of Biomass Production in Colony-Forming Green Algae, Botryosphaerella sudetica, Under Mixotrophic Cultivation.

In this study, we characterized the potential of colony-forming green algae, Botryosphaerella sudetica KNUA107, isolated from Ulleung Island, South Korea, as a bioresource and analyzed the effects of mixotrophic cultivation on its bioresource production efficiency. Internal transcribed spacer (ITS) (ITS1, 5.8S, and ITS2), ribulose bisphosphate carboxylase large subunit (rbcL), and elongation factor Tu (tufa) regions were used for molecular identification and phylogenetic analysis. B. sudetica KNUA107 had a strong relationship with the green algae of Botryococcus and Botryosphaerella genera, which are colony-forming species, and was also associated with members of the Neochloris genus. To improve biomass productivity, we tested mixotrophic cultivation conditions using several organic carbon sources. Glucose supplementation stimulated B. sudetica KNUA107 growth and reduced the time needed to reach the stationary phase. In addition, the colony size was 1.5-2.0 times larger with glucose than in photoautotrophic cultures, and settleability improved in proportion to colony size. The total lipid content and biomass productivity were also higher in cultures supplemented with glucose. Among the lipid components, saturated fatty acids and monounsaturated fatty acids had the highest proportion. Our study suggests that B. sudetica KNUA107, which has enhanced efficiency in biomass production and lipid components under mixotrophic cultivation, has high potential as a bioresource.

Environmental Factors Associated with the Eukaryotic Microbial Community and Microalgal Groups in the Mountain Marshes of South Korea.

The diversity indices of eukaryotic microalgal groups in the Jeonglyeongchi, Waegok, and Wangdeungjae marshes of Mount Jiri, Korea, were measured using Illumina MiSeq and culture-based analyses. Waegok marsh had the highest species richness, with a Chao1 value of 828.00, and the highest levels of species diversity, with Shannon and Simpson index values of 6.36 and 0.94, respectively, while Wangdeungjae marsh had the lowest values at 2.97 and 0.75, respectively. The predominant species in all communities were Phagocata sibirica (Jeonglyeongchi, 68.64%), Aedes albopictus (Waegok, 34.77%), Chaetonotus cf. (Waegok, 24.43%), Eimeria sp. (Wangdeungjae, 26.17%), and Eumonhystera cf. (Wangdeungjae, 22.27%). Relative abundances of the microalgal groups Bacillariophyta (diatoms) and Chlorophyta (green algae) in each marsh were respectively: Jeonglyeongchi 1.38% and 0.49%, Waegok 7.0% and 0.3%, and Wangdeungjae 10.41% and 4.72%. Illumina MiSeq analyses revealed 34 types of diatoms and 13 types of green algae. Only one diatom (Nitzschia dissipata) and five green algae (Neochloris sp., Chlamydomonas sp., Chlorococcum sp., Chlorella vulgaris, Scenedesmus sp.) were identified by a culture-based analysis. Thus, Illumina MiSeq analysis can be considered an efficient tool for analyzing microbial communities. Overall, our results described the environmental factors associated with geographically isolated mountain marshes and their respective microbial and microalgal communities.

Characterization of Chlorella sorokiniana and Chlorella vulgaris fatty acid components under a wide range of light intensity and growth temperature for their use as biological resources.

This study aims to characterize the potential of three strains of microalgal species (Chlorella sorokiniana KNUA114 and KNUA122; C. vulgaris KNUA104) for use as feedstock, based on their fatty acid compositions. Each strain was molecularly identified using four marker genes (ITS, SSU, rbcL, and tufA) and phylogenetically characterized. C. sorokiniana and C. vulgaris collected from Ulleung Island, South Korea, were homologous with other known species groups. Samples' fatty acid components were measured using GC/MS analysis in growth temperatures of 10 °C, 25 °C, and 35 °C. The growth rate of C. sorokiniana strains was higher than that of C. vulgaris under high-temperature conditions, confirming the potential industrial applicability of the former as feedstock material. Additionally, saturated fatty acid contents and productivities increased as biological resources of the C. sorokiniana strains were higher than those of C. vulgaris under high light intensity and temperature conditions. These results suggest that the fatty acid components of C. sorokiniana strains may potentially be used as biological resources (e.g., feedstock).

Effects of Co-culture on Improved Productivity and Bioresource for Microalgal Biomass Using the Floc-Forming Bacteria Melaminivora Jejuensis.

Bacterial and algal floc formation was induced by inoculating three species of wastewater-derived bacteria (Melaminivora jejuensis, Comamonas flocculans, and Escherichia coli) into algal cultures (Chlorella sorokiniana). Bacterial and algal flocs formed in algal cultures inoculated with M. jejuensis and C. flocculans, and these flocs showed higher sedimentation rates than pure algal culture. The floc formed by M. jejuensis (4988.46 ± 2589.81 µm) was 10-fold larger than the floc formed by C. flocculans (488.60 ± 226.22 µm), with a three-fold higher sedimentation rate (M. jejuensis, 91.08 ± 2.32% and C. flocculans, 32.55 ± 6.33%). Biomass and lipid productivity were improved with M. jejuensis inoculation [biomass, 102.25 ± 0.35 mg/(L·day) and 57.80 ± 0.20 mg/(L·day)] compared with the productivity obtained under pure algal culture conditions [biomass, 78.00 ± 3.89 mg/(L·day) and lipids, 42.26 ± 2.11 mg/(L·day)]. Furthermore, the fatty acid composition of the biomass produced under pure algal culture conditions was mainly composed of C16:0 (43.67%) and C18:2 (45.99%), whereas the fatty acid composition of the biomass produced by M. jejuensis was mainly C16:0 (31.80%), C16:1 (24.45%), C18:1 (20.23%), and C18:2 (16.11%). These results suggest the possibility of developing an efficient method for harvesting microalgae using M. jejuensis and provide information on how to improve biomass productivity using floc-forming bacteria.

Illumina MiSeq Analysis and Comparison of Freshwater Microalgal Communities on Ulleungdo and Dokdo Islands.

Ulleungdo and Dokdo are volcanic islands with an oceanic climate located off the eastern coast of South Korea. In the present study, we used barcoded Illumina MiSeq to analyze eukaryotic microalgal genera collected from Seonginbong, the highest peak on Ulleungdo, and from groundwater sites on Dongdo and Seodo Islands, which are part of Dokdo. Species richness was significantly greater in the Seonginbong samples than in the Dongdo and Seodo samples, with 834 operational taxonomic units (OTUs) identified from Seonginbong compared with 203 OTUs and 182 OTUs from Dongdo and Seodo, respectively. Taxonomic composition analysis was also used to identify the dominant microalgal phyla at each of the three sites, with Chlorophyta (green algae) the most abundant phyla on Seonginbong and Dongdo, and Bacillariophyta (diatoms) the most abundant on Seodo. These findings suggest that differences in the abundances of Chlorophyta and Bacillariophyta species in the Seonginbong, Dongdo, and Seodo samples are due to variations in species richness and freshwater resources at each sampling location. To the best of our knowledge, this is the first report to detail freshwater microalgal communities on Ulleungdo and Dokdo. As such, the number of species identified in the Seonginbong, Dongdo, and Seodo samples might be an indicator of the ecological differences among these sites and varying characteristics of their microbial communities. Information regarding the microalgal communities also provides a basis for understanding the ecological interactions between microalgae species and other eukaryotic microorganisms.Ulleungdo and Dokdo are volcanic islands with an oceanic climate located off the eastern coast of South Korea. In the present study, we used barcoded Illumina MiSeq to analyze eukaryotic microalgal genera collected from Seonginbong, the highest peak on Ulleungdo, and from groundwater sites on Dongdo and Seodo Islands, which are part of Dokdo. Species richness was significantly greater in the Seonginbong samples than in the Dongdo and Seodo samples, with 834 operational taxonomic units (OTUs) identified from Seonginbong compared with 203 OTUs and 182 OTUs from Dongdo and Seodo, respectively. Taxonomic composition analysis was also used to identify the dominant microalgal phyla at each of the three sites, with Chlorophyta (green algae) the most abundant phyla on Seonginbong and Dongdo, and Bacillariophyta (diatoms) the most abundant on Seodo. These findings suggest that differences in the abundances of Chlorophyta and Bacillariophyta species in the Seonginbong, Dongdo, and Seodo samples are due to variations in species richness and freshwater resources at each sampling location. To the best of our knowledge, this is the first report to detail freshwater microalgal communities on Ulleungdo and Dokdo. As such, the number of species identified in the Seonginbong, Dongdo, and Seodo samples might be an indicator of the ecological differences among these sites and varying characteristics of their microbial communities. Information regarding the microalgal communities also provides a basis for understanding the ecological interactions between microalgae species and other eukaryotic microorganisms.

Prolotherapy-induced Cervical Spinal Cord Injury - A Case Report -.

A 49-year-old man received prolotherapy in the upper cervical region at a local medical clinic. Immediately after the procedure, he felt a sensation resembling an electric shock in his right upper and lower extremities, and continuously complained of numbness and discomfort in the right hemibody. He visited our clinic a week later. Upon physical examination, there were no significant abnormal findings. The visual analog scale was 60 points. T2-weight magnetic resonance images of the cervical spine showed a 0.7 cm sized bright oval spot on the right side of the spinal cord at the level of C4-C5 disc, suggesting spinal cord injury. There were no definite electrodiagnostic abnormalities. Digital infrared thermal images showed moderately decreased surface temperature on lateral aspect of the right forearm and dorsum of the right hand compared with the other side. Considering that very rare complications like spinal cord injury may develop after prolotherapy, we suggest that special interventions such as prolotherapy be performed by professional experts.

Dural arteriovenous fistula on the brain stem and upper cervical spinal cord - a case report -.

A 53-year-old man abruptly developed headache and unconsciousness. Brain computed tomography (CT) and CT angiography showed subarachnoid hemorrhage, intraventricular hemorrhage, and multiple tortuous vascular structures on the brain stem and upper cervical spinal cord. Four-vessel angiography displayed intradural ventral arteriovenous fistula, supplied by the left vertebral and occipital arteries. Drainage was via both sigmoid sinus and cervical venous plexus. He had been treated with transarterial coil embolization of the left vertebral artery. Subsequently, he suffered from left hemiplegia and cognitive problem. Brain magnetic resonance (MR) and MR angiography performed 4 weeks later revealed multiple infarctions on the left cerebellum, left upper cervical spinal cord, and both medial thalamus, as well as occlusion of the left vertebral artery with reduction in varix size. After rehabilitative management, his muscle strength and cognitive function improved. We report a very rare case of dural arteriovenous fistula on the brain stem and upper cervical spinal cord.