Determination of minimum inhibitory concentrations

In liquid culture, the growth rate of D. salina CCAP 19/18 was significantly retarded by a bleocin concentration of 0.25 mg L-1. In the presence of 0.5 and 1.0 mg bleocin L-1, however, growth was completely inhibited; yet, cells were able to survive for over a week. In accordance with this restricted cell division, these cells exhibited physical manifestations of stress including minimal chlorophyll pigmentation and reduced motility. Of most relevance to the current study, bleocin concentrations of 2.0 mg L-1 and higher proved to eventually eradicate the algal cultures, although some viable cells persisted for three to five days. Similar results were observed when D. salina was cultivated on solid medium containing comparable concentrations of bleocin; however, the rate of growth on agar plates was expectedly much slower. An appreciable number of D. salina colonies were able to survive on solid medium containing 2.0 mg bleocin L-1 for nearly one month, while concentrations of 4.0 mg bleocin L-1 and higher killed the cells within one week. Growth curves for liquid cultures that exhibited prolonged viability (0.25, 0.5, 1 mg L-1) are depicted in Figure 5. Based on these findings, the M. I.C. of bleocin for this microalgal strain were found to be 2.0 mg L-1 in liquid culture and 4.0 mg L-1 on solid medium. Both conditions of selection require at least one week of exposure to the respective M. I.C. of bleocin.

The commercial herbicide Basta®, which employs PPT as its active ingredient, proved to be the most potent and fastest-acting selective agent tested. PPT concentrations of 1 mg L-1 and higher were able to kill D. salina cells within a matter of hours. In the presence of 0.5 mg PPT L-1, the growth rate was essentially negligible and these cultures were not sustainable for more than three days. Lastly, although 0.25 mg PPT L-1 reduced the rate of cell division, cultures remained viable for over a week. This concentration of PPT also induced noticeable signs of toxicity such as inhibited motility and increased carotenoid pigmentation in the algae. On solid medium, D. salina was significantly more tolerant to PPT than in liquid culture. Cells survived on agar plates containing 0.25, 0.5 and 1.0 mg L-1 for over one month. Qualitatively, an inhibition of cell growth was reflected both in the relative number of surviving cells, which naturally was inversely proportional to PPT concentration, as well as the prolonged viability of these slower growing colonies due to their more gradual exhaustion of available nutrients. After one month, the cells were rich in |3-carotene and noticeably orange in color. Figure 6 shows a side-by-side comparison of D. salina spot tested on 1 M NaCl medium with no selective agent added (left) and 0.25 mg PPT L-1 (right). The threshold for complete cell death on solid medium was clearly crossed at a PPT concentration of 2.0 mg L-1 after one week.

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Fig. 5. Dosage response curves for D. salina CCAP 19/18.

While paromomycin is toxic to related microalgae, D. salina CCAP 19/18 was found to be remarkably insensitive to this antibiotic. In the presence of concentrations as high as 400 mg paromomycin L-1, the algal cells were able to proliferate vigorously. Consequently, paromomycin was deemed unsuitable as a selective agent for the genetic transformation of D. salina.

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Fig. 6. Stress-induced accumulation of p-carotene by D. salina.

[Bleocin] mg L-1

Growth Rate (Liquid)

Cell Viability (Solid)

4.0

x

x

3.0

x

2.0

x

+

1.0

0

+

0.5

0

+

0.25

+

0

+

+

[Phosphinothricin] mg L-1

Growth Rate (Liquid)

Cell Viability (Solid)

4.0

x

x

3.0

x

x

2.0

x

x

1.0

x

+

0.5

x

+

0.25

+

0

+

+

[Paromomycin] mg L-1

Growth Rate (Liquid)

Cell Viability (Solid)

400

+

+

300

+

+

200

+

+

100

+

+

50

+

+

25

+

+

0

+

+

Table 3. D. salina CCAP 19/18 growth response to antibiotic and herbicide exposure characterized as either normal (+), inhibited (-), or negligible (0). Concentrations that engendered death of the entire algal population within one week are denoted with an x.

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