2.1.2. Secondary screening
2.1.2.1. Ability to release 5-aminolevulinic acid (ALA). Based on the
results of primary screening, any PNSB strains that grew well in GA
medium containing 0.25% NaCl under both incubating conditions
were further tested for their ability to release ALA into culture
broths. The criterion for good growth required an OD660 unit
exceeding 1.0. It is well recognized that PNSB release more ALA into
the environment under microaerobic light conditions than under
aerobic dark conditions (Saikeur et al., 2009). Therefore,
microaerobic-light conditions were used in this part of the
study, by adding 2 mL of inoculum into a screw cap test tube
(150 15 mm: 20 mL) containing 18 mL GA with 0.25% NaCl. All
culture tubes were incubated in a shaking water bath (30 rpm) for
mixing with the temperature controlled at 30 C, for 72 h. The
growth (OD660), pH and the amount of extracellular ALA were
recorded at the start time, and then every 24 h for 72 h.
Extracellular ALA was determined by a colorimetric method
(Burnham, 1970). Each culture broth was centrifuged at 4307 g
for 15 min. Then, 1 mL of the clear supernatant was mixed with
2 mL of 1 M sodium acetate buffer, pH 4.7, and 50mL of
acetylacetone. Then the mixture was boiled for 15 min and
immediately cooled, and finally 3.5 mL of modified Ehrlich’s
reagent was added to each sample. The ALA that formed was
measured at OD 553 nm, and the amount of ALA was calculated
from a standard curve relating ALA concentrations to the OD553
values.