The following are some suggestions for avoiding difficulties with isolation. In
many cases, the target organisms form clusters within a collected sample. When the
clusters are clearly visible, an effective appropriate may be pulverization using
surfactants or ultrasound. Because some microalgae will be killed off by even a
second of treatment in an ultrasound cleaning device, one must be prepared to
sacrifice some diversity.
An efficient approach for target strains with flagella is the use of phototaxis.
Isolation can be achieved relatively easily by exposing only a portion of the culture
to light and gathered the cells that cluster there.
If no diatoms (a form of single-celled algae) are needed, it may suffice to place
approximately 5–10 mg/L germanium dioxide (GeO 2 ) in the medium. By competing with silicon dioxide (SiO 2 ), GeO 2 deters silicon absorption, resulting in an
environment where diatoms cannot grow.
It takes at least one week for single cells to divide enough to form a colony that
is visible to the unaided eye. To avoid potential negative effects from medium
evaporation in the meantime, the plate may be stored in a thin polythene bag with a
zipper. Microalgae growth can also be promoted and the isolation process hastened
by introducing small amounts of CO 2 to the bag.
Examination of the growth of the marine green alga Chlorococcum littorale
under conditions of changing CO 2 partial pressure in the liquid medium showed a
specific growth rate over ten times higher when 2% CO 2 was mixed into air (CO 2
partial pressure of 0.02) than for air alone (CO 2 partial pressure of 0.00036). The
same tendency has been observed in other microalgae, indicating that CO 2 addition
is an effective means of promoting microalgae growth.
E. Selection of Microalgae Containing Oil
Nile Red (7-diethylamino-3,4-benzophenoxazine-2-one, C 20 H 18 N 2 O 2 , 318.369 gmol
−1
)
is a lipid droplet that is widely used as a stain for plants, animals, and bacteria. The
following a simple screening method using this pigment for microalgae containing
high concentrations of oil. It can be performed with a fluorometer and does not require
a fluorescence-activated cell sorter (FACS) or other expensive equipment. The same
approach can be applied with boron dipyrromethene (BODIPY), differing only in the
wavelengths.
1. Prepare a culturing solution of sufficient turbidity.
In the case of agar plates, effective dispersion can be achieved by scratching
with a platinum loop and suspension in a 10 mM phosphoric acid buffer solution (pH 7.0). Culture medium may be used as is. The target cell concentration is
around 0.2 at an optical turbidity of 720 nm.
2. Place 3 ml of cell suspension in a cuvette with transparent sides.
3. Introduce 10 µl of Nile red ethanol solution (0.5 mg/mL) and spin while
holding entrance down with Parafilm.
4. Measure a 550–650 nm emission pattern with excitation of 488 nm.
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9 Marine Bioenergy Production
many cases, the target organisms form clusters within a collected sample. When the
clusters are clearly visible, an effective appropriate may be pulverization using
surfactants or ultrasound. Because some microalgae will be killed off by even a
second of treatment in an ultrasound cleaning device, one must be prepared to
sacrifice some diversity.
An efficient approach for target strains with flagella is the use of phototaxis.
Isolation can be achieved relatively easily by exposing only a portion of the culture
to light and gathered the cells that cluster there.
If no diatoms (a form of single-celled algae) are needed, it may suffice to place
approximately 5–10 mg/L germanium dioxide (GeO 2 ) in the medium. By competing with silicon dioxide (SiO 2 ), GeO 2 deters silicon absorption, resulting in an
environment where diatoms cannot grow.
It takes at least one week for single cells to divide enough to form a colony that
is visible to the unaided eye. To avoid potential negative effects from medium
evaporation in the meantime, the plate may be stored in a thin polythene bag with a
zipper. Microalgae growth can also be promoted and the isolation process hastened
by introducing small amounts of CO 2 to the bag.
Examination of the growth of the marine green alga Chlorococcum littorale
under conditions of changing CO 2 partial pressure in the liquid medium showed a
specific growth rate over ten times higher when 2% CO 2 was mixed into air (CO 2
partial pressure of 0.02) than for air alone (CO 2 partial pressure of 0.00036). The
same tendency has been observed in other microalgae, indicating that CO 2 addition
is an effective means of promoting microalgae growth.
E. Selection of Microalgae Containing Oil
Nile Red (7-diethylamino-3,4-benzophenoxazine-2-one, C 20 H 18 N 2 O 2 , 318.369 gmol
−1
)
is a lipid droplet that is widely used as a stain for plants, animals, and bacteria. The
following a simple screening method using this pigment for microalgae containing
high concentrations of oil. It can be performed with a fluorometer and does not require
a fluorescence-activated cell sorter (FACS) or other expensive equipment. The same
approach can be applied with boron dipyrromethene (BODIPY), differing only in the
wavelengths.
1. Prepare a culturing solution of sufficient turbidity.
In the case of agar plates, effective dispersion can be achieved by scratching
with a platinum loop and suspension in a 10 mM phosphoric acid buffer solution (pH 7.0). Culture medium may be used as is. The target cell concentration is
around 0.2 at an optical turbidity of 720 nm.
2. Place 3 ml of cell suspension in a cuvette with transparent sides.
3. Introduce 10 µl of Nile red ethanol solution (0.5 mg/mL) and spin while
holding entrance down with Parafilm.
4. Measure a 550–650 nm emission pattern with excitation of 488 nm.
336
9 Marine Bioenergy Production