applied sciences Article Spatial Variations in Microbial Compositions in a Karst Critical Zone in Samcheok, Republic of Korea Heejung Kim , Jiwook Jang , Sangwook Park , Chang-Seong Kim , Han-Sun Ryu and Jin-Yong Lee * Department of Geology, Kangwon National University, Chuncheon 24341, Korea; [email protected] (H.K.); [email protected] (J.J.); [email protected] (S.P.); [email protected] (C.-S.K.); [email protected] (H.-S.R.) * Correspondence: [email protected]; Tel.: +82-33-250-8560 Received: 16 June 2020; Accepted: 6 July 2020; Published: 8 July 2020 Abstract: The effects of the unpredictability of the flow system in a karst area, with respect to bacterial communities in the aquatic environment, were evaluated. Bacterial communities from two different types of flow conditions (spring and sinkhole) were characterized and compared in the karst area. Proteobacteria, Bacteroidetes, and Verrucomicrobia were the major phyla present in the sampled spring and sinkhole waters. The water samples from the spring points were heavily loaded with Actinobacteria, especially Firmicutes, which accounted for 4.3% of the bacterial content. Furthermore, Actinobacteria were prevalent in some water samples, serving as indicators of the effects of seawater inundation. The richness and diversity of bacterial species were evaluated at the spring and sinkhole water points. The spring waters showed a higher bacterial richness and diversity compared to the sinkhole waters. Our results provide valuable information for the evaluation and investigation of microbial compositions in karst areas, which are characterized by heterogeneous hydrological conditions. The microbial species in karst areas are already exposed to changing hydrogeological conditions, and are likely to be confronted with future changes; thus, their spatiotemporal variations reflect the shifting baselines of physicochemical and ecosystem processes. Keywords: karst hydrogeology; karst critical zone; pyrosequencing analysis; microbial compositions; microbial processes 1. Introduction Karst regions contain abundant exposures of limestone or dolomite above the erosion surface, and a well-developed groundwater system formed by the erosion of these rock bodies. A variety of distinctive karst landforms occur both on the ground surface and underground. The term karst was originally used for the limestone regions of Slovenia and Croatia in the former Yugoslavia, and it referred to desert land covered with rocks. Owing to the geological characteristics and excellent underground conduit development in limestone regions, underground seepage of rainwater is rapid, and groundwater recharge is remarkably high [1]. In addition, a karst terrain exhibits unique geological features, and has a significant effect on the water quality of rivers and groundwater due to water–rock interactions [2] (Figure1). Carbonate regions account for approximately 20% of the Earth’s surface [3], and many of these regions utilize groundwater for daily water supply [4]. Therefore, it is crucial to protect the quality and supply of groundwater in these regions. Previous research has analyzed the hydrological characteristics and flow patterns of groundwater in carbonate regions [5,6]. These characteristics are also observed in some limestone regions of Gangwon-do (Samcheok, Yeongwol, Jeongseon and Sokcho) and the northern parts of Chungcheongbuk-do (Danyang-gun) in Korea. In the carbonate areas of Korea, Appl. Sci. 2020, 10, 4714; doi:10.3390/app10144714 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, x 2 of 18 Appl. Sci. 2020, 10, 4714 2 of 17 Sokcho) and the northern parts of Chungcheongbuk-do (Danyang-gun) in Korea. In the carbonate groundwaterareas of Korea, is usedgroundwater for both agriculturalis used for purposesboth agricultural and daily purposes use, prompting and daily numerous use, prompting primary investigationsnumerous primary and impactinvestigations survey and reports impact [7]. survey However, reports academic [7]. However, research academic on the hydro-ecological research on the characteristicshydro-ecological of thecharacteristics karst regions of in Korea,the karst focusing regions on theirin Korea, hydrological, focusing hydrogeological on their hydrological, and flow characteristics,hydrogeological has and been flow minimal. characteristics, has been minimal. Figure 1. Unique hydrogeological features of a karst area. Prominent featuresfeatures of of karst karst hydrogeology hydrogeology include include diffi cultydifficulty in identifying in identifying flow paths flow and paths variable and fluctuationsvariable fluctuations in flow in rates flow [8 rates–10]. [8–10]. Furthermore, Furthermore, the hydrologic the hydrologic properties properties and and flow flow patterns patterns of groundwaterof groundwater and and surface surface water water in the in carbonatethe carbonate regions regions have have not yet not been yet clearlybeen clearly identified, identified, due to due the heterogeneousto the heterogeneous and complex and complex petrological petrological properties proper of carbonateties of carbonate rocks [11 rocks–16]. [11–16]. Therefore, Therefore, it is diffi cultit is todifficult evaluate, to evaluate, predict and predict manage and groundwatermanage groundwate flow andr flow pollutant and pollutant movement movement in these regions. in these Asregions. such, climateAs such, change, climate hydrogeology,change, hydrogeology, water resource water managementresource management and human and impacts human areimpacts all key are research all key areasresearch for areas karst landscapes.for karst landscapes. The United Nations Educational, ScientificScientific and Cultural Organization (UNESCO) International Geoscience Program (IGCP) has beenbeen promoted by the InternationalInternational Union of GeologicalGeological Sciences (IUGS) since 1972. The The program program conducts conducts international international research research in in five five areas, areas, namely, namely, groundwater, groundwater, climate change,change, geodynamics,geodynamics, geological geological resources resources and and geological geological disasters. disasters. Recently, Recently, the the IGCP IGCP 661 661 has formedhas formed an international an international collaborative collaborative research research team to team conduct to conduct a global comparativea global comparative project regarding project theregarding structure, the materialstructure, circulation material circulation and environmental and environmental sustainability sustainability in key areas in key of the areas karst of the systems. karst Tosystems. date, IGCPTo date, 661 IGCP has analyzed 661 has analyzed the influence the influenc of the geologicale of the geological structure, structure, and the impactand the ofimpact climate of andclimate vegetation and vegetation differentiation, differentiation, on the type on of the critical type zone. of critical It has also zone. analyzed It hasthe also hydrogeological analyzed the structurehydrogeological and characteristics structure and of, characteristics the biogeochemical of, th processe biogeochemical in, and the process effect of in, microbial and the action effect on,of themicrobial material action cycle on, of the criticalmaterial zone. cycle Additionally, of the critical it zone. has evaluated Additionally, the water it has ecological evaluated and the carbon water uptakeecological functions, and carbon as well uptake as the functi impactons, factors, as well record as the index,impact and factor mechanismss, record index, for maintaining and mechanisms critical zones,for maintaining and has established critical zones, monitoring and has stationsestablished and monitoring standards forstations several and key standards karst regions for several across key the worldkarst regions [17–22]. across the world [17–22]. The InternationalInternational AssociationAssociation ofof Hydrological Hydrological Sciences Sciences (IAH), (IAH), founded founded in in 1971 1971 and and consisting consisting of 4500of 4500 members members from from 81 countries 81 countries around around the world, the formedworld, theformed IAH Commissionthe IAH Commission on Karst Hydrology on Karst Hydrology to actively engage in research activity and exchange [23–28]. The IAH Commission Appl. Sci. 2020, 10, 4714 3 of 17 to actively engage in research activity and exchange [23–28]. The IAH Commission promotes the development of scientific foundations and management technologies for the management and protection of water resources in karst regions, to ensure the reliable supply of drinking and agricultural water. Participants include eminent hydrogeologists from countries with karst terrains, such as China, the United States, Canada and European countries, and promote active research exchange through annual research seminars and workshops. However, relatively little research has been conducted on the water chemistry of groundwater and river water in limestone areas; specifically, studies on the microbial communities inhabiting karst water environments are scarce [29]. Therefore, as these regions have a substantial influence on water quality and microbial community composition due to the geological properties of carbonate rocks, basic analyses of river–groundwater–microbial community interactions and biogeochemical processes are required in order to ensure the reliability of water resources in karst regions. Such research should include integrated hydrogeological and hydro-ecological studies. In this study, we evaluate