Analysis of total bacterial number in seawater
The total number of bacteria in S9905 seawater was analyzed by DAPI staining and counting during 7 days. The iron content in S9905 seawater was determined to be 7.8 nM by ICP analysis. Seawater was amended with trace amount of DEF (1 nM) plus 3OC6-HSL (1 nM) or C8-HSL (1 nM), DEF(1 nM) only, each of HSL(1 nM) only. Total bacterial numbers were shown to increase after the addition of DEF, HSL or DEF plus an HSL in comparison with those obtained from seawater without any addition (Fig. 1A). However, the total bacterial number in the seawater with the addition of DEF or HSL only was observed to reach to a maximum value and then began to decrease after the sixth day (Fig. 1A), after which low amounts were recorded during the remaining time of the four-week incubation period (data not shown). Total bacterial numbers in seawater treated with DEF plus an HSL increased during seven days and the value was found to start decreasing after day 14 during four-weeks incubation period (data not shown). Comparing the maximum values of the total bacterial numbers during the first week, the values for DEF, 3OC6-HSL, C8-HSL, DEF plus 3OC6-HSL, and DEF plus C8-HSL treated seawater were 2.50-fold, 2.69-fold, 1.60-fold 3.14-fold, and 2.62-fold respectively higher than plain seawater. The increase in total bacterial number in the seawater control was unclear but such bacterial changes in bacterial abundance have also been observed in several iron enrichment experiments [5,21].
To confirm the effects of such chemical compounds as a nutrient or not, high concentrations of DEF, HSL, DEF plus HSL were amended to the same seawater, and the total bacterial number was determined by DAPI counting (Fig. 1B). The total bacterial number was also stimulated by the addition of high concentrations of DEF, HSL, DEF plus HSL. The maximum value of S9905 seawater was 5.07 × 105 cells/ml, and 4.22 × 105 cells/ml for 1 μM of DEF plus 1 μM of 3OC6-HSL, and 1 μM of DEF plus 1 μM of C8-HSL amended seawater samples respectively, which was similar to 0.1 nM of DEF plus 0.1 nM of 3OC6-HSL (4.72 × 105 cells/ml), and 0.1 nM of DEF plus 0.1 nM of C8-HSL (3.95 × 105 cells/ml) amended seawater samples. Also, the trend of bacterial number increase was very similar for the seawater with the amended with HSL under high or low concentration. However, the maximum value of S9905 1 μM of DEF amended seawater was 1.42 × 105 cells/ml, which was lower than the control seawater (Fig. 1B).
Repeat experiments were performed with S0011 seawater, which was collected from a different location. The iron content in S0011 seawater was 11.2 nM and total bacterial number was shown in Fig. 2. S0011 seawater was observed to contain more bacteria (2.1 × 105 cells/ml) than that in S9905 seawater (2.07 × 104 cells/ml). The total bacteria number in 0.1 nM DEF plus 3OC6-HSL or C8-HSL (Fig 2A) showed a similar pattern with that in Fig 1A, where an increasing trend in 7 days incubation occurred except for a drop in values for 2 nd-day samples. Comparing the maximum values of the total bacterial numbers, the values for DEF, 3OC6-HSL, C8-HSL, DEF plus 3OC6-HSL, and DEF plus C8-HSL treated seawater were 3.64-fold, 2.73-fold, 2.73-fold, 4.84-fold, and 7.92-fold respectively higher than plain seawater. In the meantime, the bacterial counting were performed for high concentration amendments S0011 seawater (Fig. 2B), 3.80-fold, 3.66-fold 5.11-fold, and 8.18-fold respectively of bacterial number increase were observed in 1 μM of 3OC6-HSL, 1 μM of C8-HSL, 1 μM of DEF plus 1 μM of 3OC6-HSL, 1 μM of DEF plus 1 μM of C8-HSL treated seawater. There was not a large difference in the maximum values in the low (0.1 nM) and high concentration (1 μM) DEF and HSL amended seawater, but a slight change of bacterial number was also observed from 1 μM of DEF treated seawater.