Environmental Engineering Reference
In-Depth Information
Table 2
Identifi cation of
Bacillus
strains
Bacteria identifi cation methods
Biolog system
Bacillus
species
16S rRNA
FAME
T-1
B. subtilis
/
atrophaeus
B. subtilis & B. licheniformis
B. subtilis
T
′
-1
B. subtilis ss spizizenii
B. subtilis & B. amyloliquefaciens
B. amyloliquefaciens
I
′
-1a
B. licheniformis
B. subtilis
B. subtilis
identifi ed as
Bacillus
genus. 16S rDNA gene
sequencing could not clearly assign them to any
species of
Bacillus
, as both isolates showed >99 %
similarity to two distinct species (
B. subtilis
and
B. licheniformis
for T-1 and
B. subtilis
and
B.
amyloliquefaciens
for T
that bacilli are a closely related group and are
considered diffi cult to identify with metabolic
profi les, as reported by other authors (Baillie
et al.
1995
; Guinebretiere et al.
2001
).
As shown above, the 16S rDNA gene
sequence, and metabolic and fatty acid profi le
analyses are often insuffi cient for accurate dis-
crimination between
Bacillus
strains. Therefore,
Chun and Bae (
2000
) proposed the use of a partial
gyrA sequence, coding for DNA gyrase subunit
A, as a useful marker for the determination of
phylogenetic relationships. Moreover, it allows
accurate classifi cation of
B. subtilis
and related
taxa, including
B. licheniformis
,
B. mojavensis
,
B. amyloliquefaciens
, and
B. atrophaeus
. Abella
et al. (
2000
) verifi ed the feasibility of using
fl uorescein-labeled trinucleotides as “quantitative
probes” for the identifi cation of bacilli species.
Labeled TTT, GGG, and ATA triplets in 16S
rRNA sequences were hybridized to the ribonu-
cleic acid of
Bacillus
spp. whole cells and the
number of the triplets was quantifi ed by synchro-
nous fl uorescence spectrometry.
A new technique currently being studied for
identifi cation of bacilli strains is Raman spectros-
copy. This technique provides complex informa-
tion about chemical cell components, such as
carbohydrates, fatty acids, proteins, and nucleic
acids, and hence combines the discriminatory
abilities of various phenotypic characteristics
(Hutsebaut et al.
2006
). As shown, Raman spec-
troscopy enabled the unambiguous identifi cation
of at least 90 % of tested strains.
-1).
The analysis of 16S rDNA sequences is pow-
erful tool for the classifi cation of micro-
organisms; however, the presence of highly
conserved sequences in this gene sometimes dis-
enables the distinction among closely related
Bacillus
species and sub-species (Guinebretiere
et al.
2001
; Cho et al.
2004
; Wu et al.
2006
;
Miranda et al.
2008
). Another taxonomic marker
used for identifi cation of micro-organisms was
fatty acid methyl esters (FAMEs). The applica-
tion of FAME analysis for differentiation of
Bacillus
strains and identifi ed by the 16S rDNA
method did not bring a clear differentiation
between T-1 and I
′
-1a strains (Table
2
). This is
because the genus
Bacillus
consists of the
Bacillus cereus
and the
Bacillus subtilis
groups,
containing the species with almost indistinguish-
able fatty acid patterns, and the studied strains
belonged to one of them.
The last method used to clarify an ambiguous
16S rDNA and FAME identifi cation of three
Bacillus
isolates was the Biolog system. The
metabolic profi le of 94 biochemical tests as mea-
sured by the BIOLOG™ system, showed identifi -
cation matches for all three
Bacillus
spp. The T-1
strain was assigned to the species
B. subtilis
or
B.
atrophaeus
with a SIM value of 0.593 and 0.549,
respectively, based on its utilization pattern of 37
substrates. The two strains T
′
-1a were
identifi ed as
B. subtilis ss spizizenii
, with a SIM
value of 0.567 and utilizing 68 substrates, while
B. licheniformis
, with a SIM value of 0.562 utilized
50 substrates, respectively (Table
2
). Nevertheless,
the results obtained did not permit the unequivocal
identifi cation of the analysed strain and confi rmed
′
-1 and I
′
4
Production of Biosurfactants
by
Bacillus
Strains
Many strains from genus
Bacillus
have the ability
to produce biological surface active compounds
(biosurfactants) belonging to the LP group. This
Search WWH ::
Custom Search