Lake Soyang Research Articles

Cultivation of Dominant Freshwater Bacterioplankton Lineages Using a High-Throughput Dilution-to-Extinction Culturing Approach Over a 1-Year Period.

Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC (e.g., the acI lineage); however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3–20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.

High-throughput cultivation based on dilution-to-extinction with catalase supplementation and a case study of cultivating acI bacteria from Lake Soyang.

Multi-omics approaches, including metagenomics and single-cell amplified genomics, have revolutionized our understanding of the hidden diversity and function of microbes in nature. Even in the omics age, cultivation is an essential discipline in microbial ecology since microbial cultures are necessary to assess the validity of an in silico prediction about the microbial metabolism and to isolate viruses infecting bacteria and archaea. However, the ecophysiological characteristics of predominant freshwater bacterial lineages remain largely unknown due to the scarcity of cultured representatives. In an ongoing effort to cultivate the uncultured majority of freshwater bacteria, the most abundant freshwater Actinobacteria acI clade has recently been cultivated from Lake Soyang through catalase-supplemented high-throughput cultivation based on dilution-to-extinction. This method involves physical isolation of target microbes from mixed populations, culture media simulating natural habitats, and removal of toxic compounds. In this protocol, we describe detailed procedures for isolating freshwater oligotrophic microbes, as well as the essence of the dilution-to-extinction culturing. As a case study employing the catalase-supplemented dilution-to-extinction protocol, we also report a cultivation trial using a water sample collected from Lake Soyang. Of the 480 cultivation wells inoculated with a single lake-water sample, 75 new acI strains belonging to 8 acI tribes (acI-A1, A2, A4, A5, A6, A7, B1, B4, C1, and C2) were cultivated, and each representative strain per subclade could be revived from glycerol stocks. These cultivation results demonstrate that the protocol described in this study is efficient in isolating freshwater bacterioplankton harboring streamlined genomes.

Viral metagenomes of Lake Soyang, the largest freshwater lake in South Korea

A high number of viral metagenomes have revealed countless genomes of putative bacteriophages that have not yet been identified due to limitations in bacteriophage cultures. However, most virome studies have been focused on marine or gut environments, thereby leaving the viral community structure of freshwater lakes unclear. Because the lakes located around the globe have independent ecosystems with unique characteristics, viral community structures are also distinctive but comparable. Here, we present data on viral metagenomes that were seasonally collected at a depth of 1 m from Lake Soyang, the largest freshwater reservoir in South Korea. Through shotgun metagenome sequencing using the Illumina MiSeq platform, 3.08 to 5.54-Gbps of reads per virome were obtained. To predict the viral genome sequences within Lake Soyang, contigs were constructed and 648 to 1,004 putative viral contigs were obtained per sample. We expect that both viral metagenome reads and viral contigs would contribute in comparing and understanding of viral communities among different freshwater lakes depending on seasonal changes.

Long-Term Monitoring of Noxious Bacteria for Construction of Assurance Management System of Water Resources in Natural Status of the Republic of Korea.

Climate change is expected to affect not only availability and quality of water, the valuable resource of human life on Earth, but also ultimately public health issue. A six-year monitoring (total 20 times) of Escherichia coli O157, Salmonella enterica, Legionella pneumophila, Shigella sonnei, Campylobacter jejuni, and Vibrio cholerae was conducted at five raw water sampling sites including two lakes, Hyundo region (Geum River) and two locations near Water Intake Plants of Han River (Guui region) and Nakdong River (Moolgeum region). A total 100 samples of 40 L water were tested. Most of the targeted bacteria were found in 77% of the samples and at least one of the target bacteria was detected (65%). Among all the detected bacteria, E. coli O157 were the most prevalent with a detection frequency of 22%, while S. sonnei was the least prevalent with a detection frequency of 2%. Nearly all the bacteria (except for S. sonnei) were present in samples from Lake Soyang, Lake Juam, and the Moolgeum region in Nakdong River, while C. jejuni was detected in those from the Guui region in Han River. During the six-year sampling period, individual targeted noxious bacteria in water samples exhibited seasonal patterns in their occurrence that were different from the indicator bacteria levels in the water samples. The fact that they were detected in the five Korea's representative water environments make it necessary to establish the chemical and biological analysis for noxious bacteria and sophisticated management systems in response to climate change.

Monitoring of Lake area Change and Drought using Landsat Images and the Artificial Neural Network Method in Lake Soyang, Chuncheon, Korea

Monitoring of Lake area Change and Drought using Landsat Images and the Artificial Neural Network Method in Lake Soyang, Chuncheon, Korea

Numerical Simulations on the Application of a Closed-Loop Lake Water Heat Pump System in the Lake Soyang, Korea

A lake is one of the geothermal energy sources to meet increasing demands for renewable energy use. In this study, a series of numerical modeling was performed to evaluate the applicability of a close-loop lake water heat pump (LWHP) system in Lake Soyang, Korea. A non-isothermal pipe flow model was used to simulate the flow and heat transfer processes occurring in the LWHP system with the main pipe and several helical tubes for heat exchange. Based on the temperature data measured in the Lake Soyang for 4 years, the installation depth and the number of helical tubes were determined sequentially, and the sensitivities of additional installation and operation factors on the system performance were analyzed. Assuming a mild current in the lake, the installation and operation conditions for the efficient operation of the system were suggested as follows: The installation of 16 helical tubes at 50 m deep, the circulation rates of heat-carrier fluid of 189.3 L/min, the inner diameter of tubes of 32 mm, and the wall thickness and thermal conductivity of 2.9 mm and 0.4 W/mK, respectively. Considering many lakes and reservoirs in Korea, the closed-loop LWHP system would be a viable renewable energy application.

Rhodoferax lacus sp. nov., isolated from a large freshwater lake

A Gram-staining-negative, aerobic, motile with a single polar flagellum and rod-shaped bacterium as a bacterial host of podovirus P26218, designated IMCC26218T, was isolated from Lake Soyang, South Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain IMCC26218T belonged to the genus Rhodoferax of the family Comamonadaceae and shared 97.7-99.0 % sequence similarities with Rhodoferax species. The draft whole genome sequence of strain IMCC26218T was ca. 4.9 Mbp in size with the DNA G+C content of 62.3 mol%. Average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values between strain IMCC26218T and other Rhodoferax were 74.0-77.3 % and 19.5-21.0 %, respectively, showing that the strain represents a new Rhodoferax species. The strain contained summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and C16 : 0 as the major fatty acids and phosphatidylethanolamine, three unidentified phospholipids, two unidentified aminolipids and two unidentified lipids as major polar lipids. The predominant isoprenoid quinone of the strain was ubiquinone-8 (Q-8). On the basis of the phylogenetic and phenotypic characteristics, strain IMCC26218T is considered to represent a novel species of the genus Rhodoferax, for which the name Rhodoferax lacus sp. nov. is proposed. The type strain is IMCC26218T (=KACC 18983T=NBRC 112709T).

Seasonal Succession of Zooplankton Community in a Large Reservoir of Summer Monsoon Region (Lake Soyang).

Seasonal Succession of Zooplankton Community in a Large Reservoir of Summer Monsoon Region (Lake Soyang).

Multiple Linear Regression Models for Predicting Nonpoint-Source Pollutant Discharge from a Highland Agricultural Region

Sediment runoff from dense highland field areas greatly affects the quality of downstream lakes and drinking water sources. In this study, multiple linear regression (MLR) models were built to predict diffuse pollutant discharge using the environmental parameters of a basin. Explanatory variables that influence the sediment and pollutant discharge can be identified with the model, and such research could play an important role in limiting sediment erosion in the dense highland field area. Pollutant load per event, event mean concentration (EMC), and pollutant load per area were estimated from stormwater survey data from the Lake Soyang basin. During the wet season, heavy rains cause large amounts of suspended sediment and the occurrence of such rains is increasing due to climate change. The explanatory variables used in the MLR models are the percentage of fields, subbasin area, and mean slope of subbasin as topographic parameters, and the number of preceding dry days, rainfall intensity, rainfall depth, and rainfall duration as rainfall parameters. In the MLR modeling process, four types of regression equations with and without log transformation of the explanatory and response variables were examined to identify the best performing regression model. The performance of the MLR models was evaluated using the coefficient of determination (R2), root mean square error (RMSE), coefficient of variation of the root mean square error (CV(RMSE)), the ratio of the RMSE to the standard deviation of the observed data (RSR) and the Nash–Sutcliffe model efficiency (NSE). The performance of the MLR models of pollutant load except total nitrogen (TN) was good under the condition of RSR, and satisfactory for the NSE and R2. In the EMC and load/area models, the performance for suspended solids (SS) and total phosphorus (TP) was good for the RSR, and satisfactory for the NSE and R2. The standardized coefficients for the models were analyzed to identify the influential explanatory variables in the models. In the final performance evaluation, the results of jackknife validation indicate that the MLR models are robust.

Origin and fate of nitrate runoff in an agricultural catchment: Haean, South Korea – Comparison of two extremely different monsoon seasons

The monsoon season in South Korea has great influences on biogeochemical and hydrological processes in the entire country, but is specifically of concern in the Soyang lake watershed, the main drinking water reservoir for the 20-million-people metropolis Seoul. Therefore, water quality and nitrate concentration control in Lake Soyang is of high public priority. The Haean catchment is the most prominent agriculture-dominated sub-catchment of the Soyang lake watershed. It is a complex terrain influenced by extreme rain events and non-point nitrate sources. In this investigation we used input-output calculations and a stable isotope approach to quantify and determinate the origin of nitrate inputs into the rivers that later flow into the lake. During pre-monsoon and monsoon seasons in 2013 and 2014 we measured daily rainfall and river water discharge within the Haean catchment and collected rain, river water and groundwater samples in order to analyze nitrate concentrations and nitrate nitrogen and oxygen isotope abundances. Furthermore, we collected nitrogen fertilizers as applied in the catchment. Heavy monsoon events, as in 2013, were the most pronounced drivers of nitrate leaching being responsible for >80% of the nitrate output in the river runoff. On the other hand, an almost missing summer monsoon in 2014 drove the nitrate runoff in a different manner, being responsible for only 0.4% of the total nitrate nitrogen river discharge in the previous year. Results of nitrate nitrogen and oxygen isotope abundance analyses suggest soil microbial nitrification as the most important contributor to the nitrate in the river runoff. In addition, nitrate from groundwater partially affected by microbial denitrification contributed to the nitrate in the runoff due to river-aquifer exchange fluxes during the monsoon season. Direct leaching of nitrate from mineral fertilizers and atmospheric nitrate deposition were obviously only minor contributors to the nitrate in the runoff.

Assessing resource-use efficiency of land use

We introduce an explicit indicator and the Land Use Management Support System to assess the resource-use efficiency of land use (RUE) at the landscape scale. To estimate RUE, we relate land-use performance with regard to ecosystem services indicators to the maximum possible land-use performance based on an optimised land-use configuration. The test application of the RUE assessment in the Haean catchment, South Korea, shows that the land-use system's RUE could be increased by 11% for both nitrate and sediment loss. The estimated headroom could indicate whether potential contaminant reduction targets for the downstream water reservoir Lake Soyang could be achieved with the current land-use system. The recurring RUE assessment for a given region might indicate the effectiveness of spatial planning and policy measures to improve the RUE in that region. Future work should address the integration of RUE into a participatory spatial planning or resource-management framework.

Genomic and ecological study of two distinctive freshwater bacteriophages infecting a Comamonadaceae bacterium

Bacteriophages of freshwater environments have not been well studied despite their numerical dominance and ecological importance. Currently, very few phages have been isolated for many abundant freshwater bacterial groups, especially for the family Comamonadaceae that is found ubiquitously in freshwater habitats. In this study, we report two novel phages, P26059A and P26059B, that were isolated from Lake Soyang in South Korea, and lytically infected bacterial strain IMCC26059, a member of the family Comamonadaceae. Morphological observations revealed that phages P26059A and P26059B belonged to the family Siphoviridae and Podoviridae, respectively. Of 12 bacterial strains tested, the two phages infected strain IMCC26059 only, showing a very narrow host range. The genomes of the two phages were different in length and highly distinct from each other with little sequence similarity. A comparison of the phage genome sequences and freshwater viral metagenomes showed that the phage populations represented by P26059A and P26059B exist in the environment with different distribution patterns. Presence of the phages in Lake Soyang and Lake Michigan also indicated a consistent lytic infection of the Comamonadaceae bacterium, which might control the population size of this bacterial group. Taken together, although the two phages shared a host strain, they showed completely distinctive characteristics from each other in morphological, genomic, and ecological analyses. Considering the abundance of the family Comamonadaceae in freshwater habitats and the rarity of phage isolates infecting this family, the two phages and their genomes in this study would be valuable resources for freshwater virus research.

소양호 동물플랑크톤 군집의 장기변동과 환경요인: 2003~2014

소양호 동물플랑크톤 군집의 장기변동과 환경요인: 2003~2014

Water quality in Lake Soyang watershed affected by sediment runoff from a highland agricultural region

Water quality in Lake Soyang watershed affected by sediment runoff from a highland agricultural region

Genome characteristics and environmental distribution of the first phage that infects the LD28 clade, afreshwater methylotrophic bacterial group.

Bacteriophages infecting major groups of freshwater heterotrophic bacteria have been rarely isolated, hampering analyses of freshwater viromes. Here, we report the isolation and genomic characterization of P19250A, the first phage that infects the LD28 clade, an abundant freshwater methylotrophic bacterial group. P19250A was isolated from Lake Soyang, an oligotrophic reservoir, using an LD28 strain as a host. Morphological and genomic analyses revealed that P19250A is a lytic siphovirus with a ∼38.6-kb genome. To analyze the distribution of P19250A genome within its habitat, six seasonal viral metagenome (virome) samples were prepared from Lake Soyang. Through binning analysis of freshwater viromes, P19250A was shown to be the most highly assigned freshwater phage that infects heterotrophic bacteria (up to 8.21%) in five viromes. Furthermore, when freshwater virome data collected worldwide were analyzed, P19250A genome also showed high abundance, especially in Lough Neagh, UK, where P19250A genome was recorded as the most abundant bacteriophage. From metagenome analysis, the proportion of P19250A-assigned reads showed seasonal fluctuation following the abundance of the LD28 clade in Lake Soyang. These results showed that P19250A would be an essential resource for analyses of freshwater viromes, and also suggest that phages of other abundant freshwater bacteria need to be isolated for better understanding of freshwater viruses.

Stormwater Runoff Characteristics and Effective Management of Nonpoint Source Pollutants from a Highland Agricultural Region in the Lake Soyang Watershed

The dense highland field area in the upstream region of the Lake Soyang watershed is subject to excessive soil erosion during the wet season. In this study, stormwater runoff from the Lake Soyang watershed was monitored during four rainfall events at 10 locations throughout 2016. The maximum SS concentration at Naedongcheon, which is located in the upper part of the Soyang River, reached 4598 mg/L. The event mean concentration (EMC) of SS loads in Naedongcheon ranged from 82.2 mg/L to 926.3 mg/L. We found that, although the first flush events were usually concentrated in highly paved urban areas, a first flush occurred in the agricultural area of the dense highland field region. The first flush phenomenon was identified by a dimensionless cumulative runoff mass and volume curve (M(V) curve), and the intensity of the first flush was analyzed by the coefficient of the nonlinear regression model and the FF30 and FF25 values (the fraction of pollution load transported by the first 30% and 25% of runoff, respectively). Nonlinear regression models using the power function were applied to fit the M(V) curve, the FF30 values were inversely proportional to the coefficient a of the regression model. A long-term seasonal trend decomposition for monthly turbidity and precipitation was performed for the Lake Soyang. Long-term turbidity trend was approximately coincident with the trend in long-term precipitation. In addition, the present status of the best management practices (BMPs) in the upper part of the Soyang River basin was investigated, and a survey of the management and operation of the BMPs was conducted for selected farmers.

Flavobacterium chuncheonense sp. nov. and Flavobacterium luteum sp. nov., isolated from a freshwater lake.

Two Gram-staining-negative, orange coloured, non-motile, rod-shaped bacterial strains, designated strains IMCC26013T and IMCC26026T, were isolated from a freshwater sample collected from Lake Soyang in Korea. The 16S rRNA gene-based phylogenetic analyses showed that both strains belonged to the genus Flavobacterium and that strains IMCC26013T and IMCC26026T were most closely related to Flavobacterium psychrophilum (96.5 %) and Flavobacterium myungsuense (97.7 %), respectively. DNA G+C contents of strains IMCC26013T and IMCC26026T were 37.8 and 33.7 mol%, respectively. DNA-DNA relatedness between strain IMCC26026T and F. myungsuense HMD1033T was 56.4 %, showing a novel species status of strain IMCC26026T. Major fatty acid constituents (>10 %) of strain IMCC26013T were iso-C15 : 1 G, C15 : 1 ω6c, C17 : 1 ω6c and summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and those of strain IMCC26026T were iso-C15 : 0, anteiso-C15 : 0 and summed feature 3. Respiratory quinone detected in the two strains was MK-6. Both strains contained phosphatidylethanolamine as a major polar lipid. On the basis of these results, strains IMCC26013T and IMCC26026T were considered to represent novel species in the genus Flavobacterium, for which the names Flavobacterium chuncheonense (type strain IMCC26013T=KCTC 52573T=NBRC 112526T), and Flavobacterium luteum (type strain IMCC26026T=KCTC 52572T=NBRC 112527T) are proposed, respectively.

Occurrence of exotic eels in natural waters of South Korea

ABSTRACTWe surveyed the proportion of anguillid eel species inhabiting South Korea natural waters. From September 2014 to August 2015, 429 eels were collected in various habitats for identification using morphological features and DNA-based molecular methods. We found 424 Japanese eels (Anguilla japonica, 98.8%), two European eels (A. anguilla, 0.5%), one American eel (A. rostrata, 0.23%), one tropical eel (A. marmorata, 0.23%), and one short-finned eel (A. bicolor pacifica, 0.23%). Three (A. anguilla, A. rostrata, and A. bicolor pacifica) are exotic species to the natural waters of South Korea; this study is the first record of their distribution in this region. Specifically, A. anguilla was found in the Lake Soyang and Cheongpyeong, while A. rostrata was found only in the Lake Cheongpyeong, and A. bicolor pacifica was found in the Geum River estuary.

Flavobacterium soyangense sp. nov., a psychrotolerant bacterium, isolated from an oligotrophic freshwater lake.

A bacterial strain, designated IMCC26223T, was isolated from an oligotrophic freshwater lake, Lake Soyang, Korea. Cells of strain IMCC26223T were Gram-staining negative, strictly aerobic, non-motile and short-rod-shaped. Growth occurred at pH 6-8 (optimum, pH 7.0), at 4-25 °C (optimum, 15 °C) and with 0-0.5 % (w/v) NaCl (optimum, 0 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain IMCC26223T was identified as a member of the genus Flavobacterium and most closely related to Flavobacterium fluvii H7T (97.6 %), Flavobacterium segetis AT1048T (97.5 %) and Flavobacterium weaverense AT1042T (97.2 %). DNA-DNA relatedness between strain IMCC26223T and F. fluvii H7T was 41.5-51.7 % in the reciprocal hybridization. Strain IMCC26223T contained MK-6 as the major respiratory quinone. The major cellular fatty acids consisted of C16 : 0, iso-C15 : 0, anteiso-C15 : 0 and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), and the polar lipids were phosphatidylethanolamine, an unidentified aminolipid and two unidentified lipids. The DNA G+C content of strain IMCC26223T was 34.5 mol%. On the basis of 16S rRNA gene phylogeny and phenotypic characterization, strain IMCC26223T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium soyangense sp. nov., is proposed. The type strain is IMCC26223T (=KCTC 52245T=JCM 31384T).

Genomic Analysis of a Freshwater Actinobacterium, "Candidatus Limnosphaera aquatica" Strain IMCC26207, Isolated from Lake Soyang.

Strain IMCC26207 was isolated from the surface layer of Lake Soyang in Korea by the dilution-to-extinction culturing method, using a liquid medium prepared with filtered and autoclaved lake water. The strain could neither be maintained in a synthetic medium other than natural freshwater medium nor grown on solid agar plates. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain IMCC26207 formed a distinct lineage in the order Acidimicrobiales of the phylum Actinobacteria. The closest relative among the previously identified bacterial taxa was "Candidatus Microthrix parvicella" with 16S rRNA gene sequence similarity of 91.7%. Here, the draft genome sequence of strain IMCC26207, a freshwater actinobacterium, is reported with the description of the genome properties and annotation summary. The draft genome consisted of 10 contigs with a total size of 3,316,799 bp and an average G+C content of 57.3%. The IMCC26207 genome was predicted to contain 2,975 protein-coding genes and 51 non-coding RNA genes, including 45 tRNA genes. Approximately 76.8% of the protein coding genes could be assigned with a specific function. Annotation of the IMCC26207 genome showed several traits of adaptation to living in oligotrophic freshwater environments, such as phosphorus-limited condition. Comparative genomic analysis revealed that the genome of strain IMCC26207 was distinct from that of "Candidatus Microthrix" strains; therefore, we propose the name "Candidatus Limnosphaera aquatica" for this bacterium.