3.1. Physicochemical Composition
The temperature of the groundwater collected from the tubular wells installed at the GWHP in the study area displayed a considerably wide range (12.5–21.5 °C) due to the influence of the GWHP system. We observed a particularly distinct seasonal fluctuation, with the highest and lowest levels reached during summer (21.5 °C) and winter (12.5°), respectively, and a comparatively high water temperature detected in October 2014 and August 2015. The EC ranged from 186 μS/cm to 350 μS/cm, with the lowest and highest values obtained in May 2016 (186 μS/cm) and August 2016 (350 μS/cm), respectively. Exclusion of these two values resulted in a relatively stable range (204–254 μS/cm) (Figure 2
). The pH ranged from 4.4 to 8.3, although exclusion of values obtained in July 2016 returned a range of 6.0 to 8.3, with no seasonal fluctuation in pH observed. The DO ranged from 1.42 mg/L to 5.88 mg/L, with relatively high values (4.10 mg/L) found exclusively in the summer during in-flow of river water, whereas other seasons showed similar values (2.23 mg/L).
We observed a marked increase in EC in August and November of 2015, which was attributed to the inefficient circulation of groundwater at the geothermal wells that caused different EC between the upper and lower levels of groundwater [6
]. The main chemical composition of the collected samples is presented as a Piper diagram (Figure 3
a). The overall geochemical characteristics of the groundwater in the study area identified it as Ca–HCO3
-type groundwater with relatively low depth. Although some samples indicated an increased contribution of Na+
, the majority of samples showed steady values for contributions by the main cations and anions. A similar pattern to that observed in the Piper diagram was observed in a Durov diagram (Figure 3
b). In agreement with the Piper diagram, groundwater quality did not show significant compositional changes with respect to the anions; however, an increase in the cations Na+
was identified in the Durov diagram. The Ca–HCO3
classification presented by the Piper and Durov diagrams generally indicates the type of natural groundwater quality relatively less affected by pollution [33
]. Furthermore, the (total dissolved solid) value ranged from 106.4 mg/L to 134.1 mg/L. Along with the increasing trend in TDS, we observed increasing trends for Ca2+
, and HCO3−
contents, with the most distinct trends exhibited by Ca2+
, and HCO3−
, and no clear correlations identified for Na+
, and NO3−
). These findings indicated that changes in hydrogeochemical properties according to the GWHP operation were not distinct. Nevertheless, geophysicochemical analysis should continue to be conducted, as such analyses often provide basic data for use in developing response measures necessary for the preservation and management of surrounding groundwater quality required for system operation.
3.3. Identified Bacteria
The results of bacterial identification are presented in Table 2
. The Anoxybacillus
genus reportedly includes 23 species and subspecies, with Anoxybacillus amylolyticus
representing the type species. The major sites of isolation included hot springs, fertilizers, and geothermal power plants [36
]. The Anoxybacillus
genus comprises rod-shaped, Gram-positive bacteria (size: 0.4–0.9 × 2.5–5.0 µm) frequently arranged as a pair or chain, with a single pore per cell. The oxygen demand and catalase reaction varied, with some members being anaerobic and others facultatively anaerobic. The bacteria were either alkalophilic or alkalophobic, and the DNA G+C content ranged from 42% to 57% [37
Filippidou et al. [38
] isolated a strain of Anoxybacillus
from deposits clogging ground surface filters at geothermal power plants in a field of enhanced geothermal systems in the Groß Schönebeck region of northern Germany, which formed endospores that are thermophilic, Gram-positive, facultatively anaerobic, and positive for catalase and oxidase reactions. Additionally, Dai et al. [39
] isolated a thermophilic and ethanol-resistant strain of Anoxybacillus
from deposit samples collected from groundwater wells at a hot spring in Yunnan Province in China. Schäffer et al. [40
] isolated a bacterial strain from geothermally heated soil samples collected from Yellowstone National Park in the United States, identified by Coorevits et al. [41
] as belonging to the Anoxybacillus
genus (subsequently named Anoxybacillus tepidamans
Anoxybacillus spp. are found abundantly in areas close to geothermal power plants and hot springs, and from which they are easily isolated. According to previous studies, bacteria belonging to this genus are resistant to hostile environmental conditions, such as high temperature and pH, and capable of continuously increasing colony number to ultimately promote bio-clogging. In this study, seven of 14 species of thermophilic bacteria isolated and identified from SY-3 were identified as A. tepidamans, with a sequence homology of 98.6% to 98.9%.
The strain of bacteria isolated from the SY-3 GWHP (24 November 2016) was identified as 99.4% Bacillus oceanisediminis
, which was first reported by Zhang et al. [42
]. B. oceanisediminis
is a rod-shaped (size: 0.6–0.8 × 2.0–3.0 µm), anaerobic, Gram-positive bacteria that we isolated at 45 °C, despite the optimal growth conditions requiring 37 °C. Because the strain grows within a temperature range of 4 °C to 45 °C, it is difficult to classify it as a thermophilic bacterium.
To date, 59 species of Deinococcus
have been reported, with Deinococcus radiodurans
representing the type species. In this study, the dominant culturable bacteria isolated from the SY-3 GWHP (November 9, 2015) was identified as 98.1% Deinococcus geothermalis
, which has an optimal growth temperature of 45 °C to 50 °C and is classified as a thermophilic bacterium. D. geothermalis
is particularly important to the formation of biofilms [43
]. According to Kolari et al. [43
], biofilms formed by D. geothermalis
negatively influence the product quality from paper manufacturing machines. Additionally, this species should be managed as an important microorganism capable of biofilm formation. It can reduce thermal efficiency and degrade the system integrity of machines and wells associated with GWHP systems installed in the study area.
was also isolated from the SY-3 GWHP. The Effusibacillus
genus was first reported by Watanabe et al. [44
], and little is known about the three known species. Effusibacillus
spp. are spore-forming, rod-shaped, anaerobic or facultatively anaerobic chemotrophs. The Lysobacter
genus includes 13 species and are mainly isolated from soil environments. In this study, the strain of mesophilic bacteria isolated from the SY-3 GWHP samples (13 August 2014) was identified as 97.2% Lysobacter mobilis
. The Lysobacter
genus comprises Gram-negative bacteria characterized by gliding motility and generally categorized as beneficial microorganisms. These bacteria produce enzymes that exhibit diverse functions, as well as reported sources of a novel antibiotic.
spp. includes ~200 species and subspecies of facultatively anaerobic bacteria that form endospores and originate from multiple habitats, such as soil, water, and plants. In this study, among the SY-3 regions, samples collected on 27 May 2016, and 25 August 2016, resulted in isolation of two strains identified as Paenibacillus elgii
and Paenibacillus lautus
. The first reported isolation of P. elgii
was from perilla seeds and resulted in identification of a rod-shaped bacterium with motility. P. lautus
was reclassified from Bacillus lautus
by Heyndrickx et al. [45
]. Neither of the two strains has shown strain-specific functionality. Paenibacillus
is a mesophilic bacterium that can grow in temperatures up to 45 °C, thereby classifying is as a psychrophilic bacterium.
genus, first reported by Yu et al. [46
], includes Vulcaniibacterium tengchongense
and Vulcaniibacterium thermophilum
is Gram-negative bacteria that can grow in temperatures ranging from 25 °C to 55 °C, classifying them as mesophilic bacteria.
Here, we describe collection and analysis of groundwater samples from tubular wells installed at the GWHP between 2014 and 2016 in order to investigate correlations between water temperature and microbial activity. Physicochemical data and microorganism activity (psychrophilic, mesophilic, and thermophilic bacteria) revealed microbial distribution in groundwater at the study area, with an excessively high level of thermophilic bacteria and with microbial count increasing according to increases in water temperature during the summer season. Investigation of the effects of temperature on the growth of the isolated and identified culturable bacteria identified eight species of bacteria (5 thermophilic bacteria, 1 mesophilic bacterium, and 2 psychrophilic bacteria). A. tepidamans was the most dominant thermophilic bacteria in the study area, with this genus having previously been reported as capable of creating bio-clogging deposits in wells and above-ground filters at geothermal power plants. Our experimental results showed that changes in groundwater temperature according to on-site management of the GWHP had a greater influence on the activity of thermophilic bacteria than on psychrophilic bacteria among autochthonous bacteria. Analysis of the thermal efficiency of the GWHP will benefit from studies on thermophilic bacterial cultures, and contribute to the understanding of bio-clogging phenomena that is possibly mediated by A. tepidamans. Furthermore, our results imply that quantitative and qualitative studies of autochthonous bacteria inhabiting areas near GWHPs should be conducted using next-generation sequencing to determine microbial community structures.