In order to determine whether butanol tolerance was the result
of long term adaptation to butanol,six original isolated strains (prior
to adaptation) were grown on MRS plate sin the absence of butanol
and subsequently tested for butanol tolerance. L. amylovorus strain
NE-L 0206-19, L. mucosae strains BR0713-30 and BR0713-33,and
W. confusa strainBR0216-18 were chosen for this study because
they were among the most butanol tolerant strains identified;on
long-term culture,these strains were adapted and grew in the
presence of 4%butanol to an OD600Z3.0 (Tables1and2). L. brevis
strain BR0315-L5 and Lactobacillus johnsonii strain BR0315-B4 were
chosen as representatives of less tolerant strains,since they grew to
an OD600 of Z2.0 in the presence of 3% butanol,but failed to grow
on 4%butanol(Table2).
These six strains were plated on to MRS plates in the absence of
butanol,and isolated colonies were used to directly inoculate MRS
broth containing 4%butanol(Fig.1). Only highly butanol tolerant
L. mucosae strainsBR0713-30 and BR0713-33 showed growth with in
7 days,suggesting that these strains possess genetic resistance to
butanol. Notice that the short term butanol stress test(Fig.1) differs
with the long term adaptation (Tables1and2), in (a) the initial
inoculums,from single colonies versus 5%of overnight cultures,and
b) initial butanol content from 4% butanol versus gradual transfer of
cultures from2%or3%to4%butanol.Thus it is not surprising that
the growth of the L. mucosae strains BR0713-30andBR0713-33
(highly butanol tolerant after adaptation; Tables1and2) was
relatively slow,and none of there maining strains exhibited growth
in the presence of4% butanol after 7days(Fig. 1), suggesting that
their tolerance is there sultofad aptation to butanol stress selection