## 4 Discussion
### Total Bacteria and Culturable Bacteria  
Total counts of approximately 10\({}^{6}\)\(-\)10\({}^{7}\) cells mL\({}^{-1}\) of bacteria exist in coastal seawater [33], and 10\({}^{5}\)\(-\)10\({}^{6}\) cells mL\({}^{-1}\) exist in open sea [34]. In the present study, the total counts of bacteria were 5.4 \(\times\) 10\({}^{5}\)-3.0 \(\times\) 10\({}^{6}\) cells mL\({}^{-1}\) at Stn. 1, 3.9 \(\times\) 10\({}^{5}\)-1.9 \(\times\) 10\({}^{6}\) cells mL\({}^{-1}\) at Stn. 2, and 2.3 \(\times\) 10\({}^{5}\)-4.1 \(\times\) 10\({}^{5}\) cells mL\({}^{-1}\) at Stn. 3 (Figures 6a, 7a and 8a). These results are not significant deviations from the reported values. Comparing the direct counts of FLB and PAB at each station, the FLB tended to exceed the PAB on the whole at all three stations (Figures 6a, 7a and 8a). It is speculated that PAB are attached to particles and utilize the relatively rich organic matter of detritus, whereas FLB uptake dissolved oligotrophic organic matter in seawater [35].  
In the previous studies conducted in Akkeshi Bay, the reported densities of culturable bacteria were 10\({}^{2}\)-10\({}^{4}\) CFU mL\({}^{-1}\)[30]. In the present study, the values of culturable bacteria were detected within that range (Figures 6b, 7b and 8b). In Akkeshi-ko Estuary (Stn. 1), FLB tended to be more abundant than PAB. However, the densities and/or proportions (%) of PAB tended to exceed those of FLB at Stns. 2 and 3. It is considered that heterotrophic bacteria can exist as FLB at areas with relatively higher concentrations of dissolved organic matter such as Akkeshi-ko Estuary, and that the proportion of PAB increased as the stations went offshore with less dissolved organic matter.  
A review noted that the proportions of culturable bacteria to the total counts of bacteria were 0.001\(-\)0.1% in seawater of the open ocean [36]. These proportions were reported to be higher in coastal waters [37; 38]. For example, in seawater of Maizuru Bay, Japan, the total counts of bacteria were 10\({}^{5}\)-10\({}^{6}\) cells mL\({}^{-1}\) and culturable bacteria were 10\({}^{4}\)-10\({}^{5}\) CFU mL\({}^{-1}\)[37]. In the surface waters along the Adriatic coast, the proportions of culturable bacteria to the total counts of bacteria were 0.1-22% [38].  
In other environments such as Lake Biwa, the culturable bacteria were 1.7 \(\times\) 10\({}^{3}\)-1.0 \(\times\) 10\({}^{5}\) CFU mL\({}^{-1}\) when the total bacteria were 1.3 \(\times\) 10\({}^{6}\)-1.4 \(\times\) 10\({}^{7}\) cells mL\({}^{-1}\). In the Yodo River, the only river flowing out from Lake Biwa, the total culturable bacteria were 7.1 \(\times\) 10\({}^{4}\)-1.4 \(\times\) 10\({}^{6}\) CFU mL\({}^{-1}\), and the total direct counts of bacteria were 2.5 \(\times\) 10\({}^{6}\)-4.0 \(\times\) 10\({}^{7}\) cells mL\({}^{-1}\)[39]. It is reasonable to have the highest ratio of 22.91% of culturable bacteria to total counts in the Akkeshi Bay area with high productivity (Figure 7c, PAB). The proportions of culturable bacteria to the direct counts of bacteria attached to the surface of the seagrass leaves were >20% in six of the 11 surveys (Figure 5c). Similar results (1-20%) were reported for bacteria in the biofilm on the surface of seed stems in lakes in Ohnuma Quasi-National Park, Hokkaido, Japan [40]. It thus seems that many bacteria possess high viability due to the organic matter provided by the leaves of seagrass.  
In general, most of the heterotrophic bacteria in natural environments are unculturable on agar media, and this is one of the major issues that has long been unsolved in microbiology [41]. In the present study, growth-inhibiting bacteria were cultured and isolated using ST10\({}^{-1}\) agar medium, and the bacteria were limited to those that can be cultured using this medium.