August 2007, 65-68
munity analysis because it represents an excellent approach
to c o m p r e h e n d i n g the and of a
microorganisms from nature are not yet (Giraffa
and
N e v i a n i 200 1). Ampe (1 have compared thestandard microbiological techniques, and technique that do
not depend on c u l t i v a t e d processes, to examine microbial
p o p u l a t i o n s . They found t h a t t h e c u l t u r e - i n d e p e n d e n t t e c h - nique is the most suitable to d e p i c t p o p u l a t i o n dynamics.
1991, found that amplification
gene, cloning and sequencing it represents one of
Strain which is resistant to nalidixic acid, was
subsequently
grown
in media without antibiotic supplemen- tation as control Meds), the medium with the addition dixic acid 20 and the heat-treated medium byboiling at
min
to eliminate most vegetative bacterialcells. Bacteria populations grown in different treatments were examined on the first, fifth and tenth days of cultivation. Media used for the growth were as described previously
2005).
DNA Isolation. The isolation of DNA samples was
conducted as reported previously by Ampe al. 999). Amplification and Cloning of the Am-
plification of the gene was conducted employing
5'-CAggCCTAACACATgCAAgTC-3' and
for the Bacteria Domain group amplicons were purified employing Wizard Gel and the
PCR
Clean-Up System. The purified DNA's were ligated into Easy vector (Promega, Madison, USA) and transformed intoTransformants were selected on Luria Agar
media supplemented with X-gal
(40 gene in recombination li-
brary (collection of genes in vector) were amplified again using M 13F and M 13R primer (Moffett
2000) to obtain individual genes. This step would
ensure
that the amplified I genes were fromnation not from the bacterial host gene.
Amplified Ribosomal DNA Restriction Analysis (ARDRA). The gene, amplified from recombina- tion plasmids, was digested with restriction enzymes RsaI or
to yield a specific pattern representative of the
percentage of specific patterns calculated an each day of fermentation and was depicted as a population dynamic curve.
RESULT
Growth in the Presence of Microbial Community. Refering to the obtained data, we made a cell
growth histogram depicting the growth profiles
Strain IB-I Nal-R in fermentation media with different treat- ments (Fig Fig indicates that fermentation media with a blanching treatment (Blc) did not enhance the growth of Strain compared to that of the control medium fermentation medium without any treatment). In the fermentation medium with nalidixic supplementation, the growth of IB- I Nal-R became very depressed. Although the population did increase, the sum of the cell count was as high as that in the control medium or blanching treatment. This result suggested that the pre-existing bacterial popula- tion in the media were essential for successful Nata fermen- tation and migt have positive or synergistic effect to the growth of Strain I
ARDRA Reveals Bacterial During Nata Fer-
mentation. Results of ARDRA analysis showed the exist-
ence of at least twenty two different bacterial group during
Nata fermentation Each ARDRA profile found in Nata
was calculated as a percentage to the total pro- files every day starting from the day up until eleventh day. Five profiles were considered to be unique because their presence could only be found over certain specific days of fermentation (Fig 3). Unique ARDRA profiles include pro- file 1 to 5. Profile 6 to 22 was not in a curve ing bacteria and as 1, Profile 2, The because we found them only on days and they did
Fig I growth in fermentation with different treatments media without treatment, media, = media with nalidixic supplementation the number following name o f the media indicates the day o f
The numbers following each treatment indicated the days of Nata fermentation (0.5 and days).
I I I I I I I I I
Volume I , 2007
not show significant percentage numbers of the total popu- lation (data is not presented at this article).
DISCUSSION
A. with resistance marker was em-
ployed, in the laboratory to examine the influence of some media treatment on the growth during Nata Three of media were used, me- dia treatment, with blanching for 10 minutes to eliminate as many as contaminants in the rncdia.
and media with nalidixic acid (20
suppress the growth of other bacteria sensitive to this anti-
biotic. The of contaminants was expected to he sup- pressed to give enable A. to 'outcompete' and yield of good gel. was marked for the
purpose of cell estimation when they were on me-
dia with nalidixic acid supplementation. We assumed that
-
I Nal-R could be maintained as dominant popula- tion, this isolate will grow fast and produce excellent Natagel.
The results showed Nata which produce during
the when the natural popu-
lation was with a blanching or by nalidixic acid was inferior quality corn-
pared to that of control media. Therefore, the
of foreign bacteria at the control might have a syner-
gistic effect and stimulate rapid growth of the A.
population. In traditional Nata fermentation, prepara- tion was often conducted under
Microbiol lndones 67
the did always fail in 'Bad
Nata'. might explain the bacteria in the
media preparation and also d u r i n g the fermentation process, could Nata production and might possibly show
exhibit symbiosis or essential factors required for cellulose
I n this study we define 'Good Nata' fermentation as which will a homogenous cellulose
gel with transparency; while Nata' fermentation
will frothy. thin less than soft
with white or opaque color Nata gel after 8 days of fermenta-
tion
this study. ARDRA to better under-
stand the bacterial involved in the production of 'Bad' and 'Good Nata'. This analysis on ex- traction of total DNA cultured and uncultured bacteria. Specific the Bacteria Domain could be identified by their specific profiles generated from
the of 1 gene with
or
Results of the ARDRA indicated that in a traditional Nata
process, A. seeding dur- ing a process could also h a v e or associa-
tion with other present, either cocunut water or
coconut milk that might generate a mutual effect in 'Good
Nata' production or an antagonistic effect in 'Bad Nata' pro- duction,
growth pattern shown in 3, indicated a sharp for profile or 5 during the course offer-
generating the 'Bad , this
2 ARDRA profile nf from group which during Nata numbers
above every column individual various of bacterial = molecular marker
5 6 7 8 9 I 6 7 8
Days
3 Pattern of 5 ARDRA a. Bad, and h
was very erratic, while at fermentation with a
good outcome, this type of profile was less erratic and tended to stabilize over time. Profile I did not show any difference of their population dynamics either in the 'Bad' or 'Good Nata' production. Profile 2 in 'Bad' fermentation tends to show fluctuation while in good fermentation it tends to sta- bilize with a low percentage of variability. On the other hand, profile 4 in both 'Good' and 'Bad' fermentation did not show the existence of different population dynamics.
This fermentation process showed the existence of
unique profiles, profile 3 and 5 from the Bacteria Domain. Profile 3 showed rather sharp difference between 'Bad' and 'Good' Nata in a fermentation in 'Bad Nata', this profile tend to fluctuate and rise in the final fermentation process. On the other hand, in with the 'Good Nata' the existence of this tended to be minimal or
was not visible. Profile 5 on day of the fermentation
process for the 'bad' result showed the highest percentage while on the same day this profile showed only 25% in a fermentation process until yielded the 'Good Nata'. In a
fermentation process with the 'bad' result, this pattern was
not detected on subsequent day, while for the fermentation process with a 'good' result, this pattern was still detectable in spite of its low amount (5%) on eleventh day. We con-
clude that community unique profiles represent one of the key factor which in this study can be considered essential indicators for 'Bad' or 'Good' fermentation. This analy- sis could be more dramatic if the sampling had not been limited to begin from day 5 where cellulose pellicles start to emerge.
Another unique matter is the amount of 'other' profiles
present in relative numbers and the flat spreading dur-
ing the fermentation process with the 'good' result as com-
pared to fermentation process ielding the 'bad' result (data not presented). A possible explanation for the existence of different types of bacteria at different steps represent a s y m -
biosis process, where a different set of bacteria are required different steps of to provide essential nutri- ents
to
A. for its cellulose biosynthesis. The pres-ence of these bacteria could supply otherwise deficient, but
essential, nutrients which are important for the growth of A. for 'Good Nata' production. This result also indi- cates that traditional Nata fermentation, which tends to be semi-aseptic, might be required to provide some beneficial bacterial inocula for 'Good Nata' production (Fig 1 .) since A. grew better in medium without antibiotic supple- mentation or having blanching treatment.
M i c r o b i o l
Other factors which might have an effect to
population dynamics is the change of p H of the t ion medium during the process. At the start
pH of the media is 3.9. This value droped over time until
it reached approximately pH 2.0 at the end of the fermenta- tion. This might have effect on the complexity bacte- rial community profiles.
This research was supported by Research Center for Mi-
crobial Divetsity, Faculty of Mathematics and Natural Sci-
ences, Agricultural University, and in Jakarta. This Research is part of CAS Thesis.
Ampe F, Ben-Omar N, C. C, Guyot
Polyphasic Stud) o f o f Microorganisms in Mexican Dough. Demonstrates the
Need for to investigate tradi-
tional A p p
Ampe F, A. 2001. o f Microbial Commu- nity for Sour Cassava Starch
by Gradient Gel Electrophoresis and
Quantitative J Microbiol
Fragment Polymorphism Analysis Program, a
Research Tool for Microbial Community Analysis Appl
Moffet BF, KA. Harris JA, Analysis of
rial Community Structure Using Anaerobe
