PRODUCTION Of NUTRIENT SOURCES FOR RHIZOBIUM

2.2.3 Screening and selecting of starch-utilizing yeast strains
Yeast strains (Section 2.2.1) were screened for their amylase production
capabilities. The yeast isolates were cultured by patching cells on starch agar (Appendix B5) plate
surface (two replicates), and allowed to grow for 3 days at 30°C. Amylolytic zones corresponding
to enzymatic degradation of starch were detected by staining the plates with iodine solution
(Hesseltine et al., 1963, Appendix A5). Colonies exhibiting large clear zones comparing to each
other were selected, and maintained using soluble starch agar medium (Appendix B6).
The starch hydrolysis and sugar alcohol production of by selected yeast strains was
also determined in liquid medium. Yeast isolates were cultivated in 50 ml of starch broth
(Appendix B7) containing 2% (w/v) of either cassava starch (Korat Cassava Industrial, Nakhon
Ratchasima, Thailand), or rice starch (Rongsenmechoheng Co., Ltd., Nakhon Ratchasima,
Thailand), or Khao-klong starch (Rongngan Pochanahanchainsiri Co., Ltd. Bangkok, Thailand) as
their carbon sources with shaking at 200 rpm and incubating at 30°C for 72 hours (two
replicates). Samples were collected. Three millilitres of culture medium were drawn aseptically
from the fermentation flask and centrifuged for 10 min at 4,500 rpm. The supernatant was
analyzed for reducing sugars by the DNS method (Miller, 1959) (Appendix A6). Cells were
disrupted by using 4 cycles of freezing in liquid nitrogen (-196°C) and thaw at 70°C, then
assayed for sugar and sugar alcohols by High-Performance Liquid Chromatography (HPLC).
Growth as cell numbers was determined using haemocytometer (Appendix C1). Starch hydrolysis
enzymes were determined.
� The measurement of α-amylase activity was performed in test tubes by adding 0.5
ml enzyme solution (diluted if necessary) to a 0.5 ml substrate solution containing 0.0067 M NaCl
and 1% soluble starch in 100 ml of 0.02 M phosphate buffer (pH 6.9) (two replicates). After 10
mins incubation at 30°C, the reaction was terminated by adding 0.5 ml 3,5-dinitrosalicylic acid
solution and quickly placing the reaction tubes into a boiling water bath for 5 min. The reaction
tubes were then cooled in running tap water and diluted with 4 ml doubled-distilled water. The
extent of product formation by enzyme reaction was monitored at 540 nm absorbance (A 540).
Glucose was used as a standard. One unit of α-amylase activity was defined as the amount of
enzyme required to liberate one µM of glucose/min or in terms of milligram of maltose liberated
in 1 minute at 30°C by 1 ml of the enzyme solution (Berfeld, 1951), even though, in the case of
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