Occurrence of cyanobacteria, actinomycetes, and geosmin in drinking water reservoir in Korea: a case study from an algal bloom in 2012

In 2012, a large concentration of geosmin was found in the Paldang reservoir, which is the primary source of drinking water in Seoul, Korea. In June and September 2012, we measured the concentrations of cyanobacteria and actinomycetes, and geosmin, to identify the source of geosmin in the Paldang reservoir. A total of 68 water samples were collected from two sampling sites (Sambong, Paldang), and used to analyze the correlation between cyanobacteria, actinomycetes, and geosmin. The cell density attained a maximum of 24,722 cells/mL on August 11, 2012 and geosmin occurred at a high concentration of 3,934 ng/L on August 13 in Sambong. After July 31, 2012 a rapid increase in growth and cell density occurred with a peak value of 11,568 cells/mL on August 6, 2012. At the same time, the geosmin concentration increased to 3,157 ng/L in Paldang. The number of cyanobacteria positively correlated with geosmin concentration (R1⁄4 0.84, P< 0.0001), while actinomycetes were not significantly correlated with geosmin (R1⁄4 0.01, P1⁄4 0.709). In addition, the number of actinomycetes was associated with increased turbidity (R1⁄4 0.507). Among the various water quality constituents, temperature affected cyanobacteria in the Paldang reservoir (R1⁄4 0.803). These results suggest that cyanobacteria are the main source of geosmin in the Paldang reservoir, which might be providing useful information for managing the unpleasant taste of its drinking water.


INTRODUCTION
Geosmin is an odorous compound that contributes to the occurrence of the earthy and musty odor in water, which has been known to decrease the quality of drinking water.
It is synthesized and secreted as a secondary metabolite by cyanobacteria, actinomycetes, and fungi (Wood et al. ; Jüttner & Watson ). Although geosmin is not associated with any serious health effects, consumers perceive it as being unsafe because of the unpleasant taste and odor that it causes in drinking water (Smith et al. ). The permissible limits for geosmin in drinking water in Japan and South Korea are 10 ng/L and 20 ng/L, respectively. However, the general population can recognize the taste and odor of geosmin at concentrations as low as 5-10 ng/L (Cook et al. ).
Geosmin cannot be efficiently removed using conventional treatments such as chlorine, ozone, and activated carbon (Koch et al. ; Cook et al. ; Lin et al. ). Therefore, a better understanding of the source of geosmin will lead to better in-lake management and drinking water treatment strategies designed to reduce its prevalence (Zuo et al. ).
The Paldang reservoir is the largest and most important source of drinking water in Korea, located near the capital city of Seoul. Its water storage capacity is ∼244 × 10 6 ton and it has a watershed area of 23,800 km 2 (Kim et al. ). The average depth is 6.5 m and it has an average water retention time of 5.4 days. This large reservoir serves as the principal source of drinking water for the >24 million people (48% of the Korean population) who live in this region; however, its earthy and musty flavor is a major concern of the drinking water industry in Korea. Geosmin is produced by a range of microorganisms, notably cyanobacteria and actinomycetes (Kutovaya & Watson ), and these geosmin-producing microorganisms may be present in the same water resource. However, it remains unknown which microorganism primarily contributes to the odor problem in the Paldang reservoir, and whether geosmin is the main source of the problem. Thus, it was necessary to analyze the parameters of all microorganisms.
The objectives of this study were: (i) to identify the main source of geosmin in the Paldang reservoir, which is an important drinking water reserve in Korea; and (ii) to analyze correlations between the numbers of geosmin and geosmin-producing microorganisms such as cyanobacteria and actinomycetes.

Water sampling
The Paldang reservoir has two major tributaries: the North Han River and the South Han River. Two water sampling sites in the Paldang reservoir, Sambong and Paldang, were used in the current study ( Figure 1). The water was sampled 3-4 times a week from June 29 to September 25, 2012 and 68 water samples were obtained. The water samples were collected in sterilized polyethylene bottles and stored at 4 C until subsequent analysis. Water quality constituents, including water temperature, pH, dissolved oxygen (DO), and turbidity, were measured immediately at the sampling site using multiprobe (YSI 6600, USA).

Quantification of cyanobacteria and actinomycetes
To quantify the number of cyanobacteria, samples were preserved using Lugol's iodine at a final concentration of 5% (Sherr & Sherr ), and kept in the dark until counting.
The cyanobacteria cell density was determined using a Sedgwick-Rafter counting chamber under a microscope (Eclipse 80i, Nikon Corporation, Sendai, Japan) using phase-contrast and bright field illumination. The number of cells in 50 of the 1,000 grids in the chamber was counted. To identity the relationship between cyanobacteria and geosmin, four cyanobacteria genera (Anabaena sp., Aphanizomenon sp., Microcystis sp., and Oscillatoria sp.) were selected.
To determine the cell density of actinomycetes, a 1 L of water sample was filtered through a Sartorius bottle top vacuum filter (polyethersulfone membrane, 0.2 μm). The membranes were cut into pieces, and placed on humic acid-vitamin (HA) agar (catalog no. H0663; MB Cell, CA, USA). Pure sterilized water (1 mL) was placed on the HA agar and spread using a glass triangle spreader; the cut filter was then detached from the HA agar. The samples were incubated at 28 C for 5-7 days followed by counting of colony-forming units (CFU) (Hirsch & Christensen ; Lee & Hwang ).   Actinomycetes were present at a concentration of 1.1 × 10 2 CFU/mL (± 64) in Sambong, and 1 × 10 2 CFU/mL (±89) in Paldang (Figure 2 Further study is needed to investigate whether geosmin could be detected at high concentrations, together with actinomycetes, in sediment of the Paldang reservoir.

Geosmin analysis
The results of the correlation coefficients of the four parameters analyzed in the current study are shown in Table 1 as a matrix. According to the matrix, geosmin only correlated with cyanobacteria (R ¼ 0.8188). The actinomycete parameters were dependent on turbidity, and the correlation coefficient was 0.5072 (Table 1). A previous study reported that the number of actinomycetes in the water source correlated with increased turbidity ( Jensen et al. ).
Previous studies reported that water temperature, DO, nutrient availability, and water transparency play important roles in the occurrence of cyanobacterial blooms (Chirico et al. ). In this study, water temperature, pH, and DO were monitored over a period of 3 months (Figure 4). The temperature ranged from 19-29 C, and 19-32 C, at Sambong and Paldang, respectively (Figure 4). At the two sampling sites, the temperature was maintained at ∼20 C, but increased to >25 C when the cyanobacterial concen-  could be triggered when the number of cyanobacteria cells exceeds 500 per milliliter.
An analysis of the water quality constituents revealed that temperature was correlated with cyanobacteria concen-

CONCLUSIONS
The current study investigated the occurrence of and correlations among cyanobacterial, actinomycetes, and geosmin levels during the summer and fall of 2012 in Paldang, a drinking water reservoir in South Korea. The densities of cyanobacteria were positively correlated with geosmin levels and were associated with high concentrations of geosmin. Also, the geosmin concentration was closely related to the cell density of Anabaena, which is a known geosminproducing cyanobacterium. Actinomycetes were associated with increased turbidity, but not geosmin concentration. In addition, among water quality constituents, a water temperature increase was related to cyanobacterial concentration.