Beruflich Dokumente
Kultur Dokumente
SYSTEI'v1ATIC AND
APPLIED MICROBIOLOGY
Summary
Cellulose-producing bacteria used for Nata de Coco production in the Philippines were isolated and
characterized. Two distinct wild-type strains (ITDI 2.1 and PA 2.2) were selected from thirty-eight isolates obtained. The two strains, and the rest of the cellulose producers, were established to be members
of the genus Acetobacter based on rapid phenotypic characterization. They were characterized and differentiated based on pellicle type and colony morphology, carbon-utilization pattern (Biolog assay),
amount of cellulose production and 16S rDNA sequence. The thick pellicle cellulose-producing strain
(ITDI 2.1) has a stable and consistent production of a very thick pellicle in any sugar-based, acidic medium, with a dry weight up to twenty-six times that of the thin pellicle cellulose-producing strain (PA 2.2)
after 7-8 days static batch culture. Species identification using 16S rDNA sequencing revealed that the
two strains belong to two different species of Acetobacter, Acetobacter xylinus and A. hansenii and may
be new subspecies under these species designation.
Key words: Acetobacter - cellulose - phenotype - 16s rRNA
Introduction
The genus Acetobacter is composed of Gram-negative,
rod-shaped, strictly aerobic, acidophilic bacteria able to
oxidize ethanol to acetic acid, and acetate and lactate to
carbon dioxide and water (DE LEY et al., 1984). Certain
members of the genus Acetobacter synthesize highly pure
extracellular polysaccharide called cellulose. To date,
there are three recognized species of Acetobacter, namely: A. xylinus (EUZEBY, 1997; formerly A. xylinum), A.
pasteurianus and A. hansenii (HOLT et al., 1994), that are
known to synthesize this polysaccharide (YAMADA, 1983;
GOSSELE et al., 1983; DE LEY et al., 1984). Cellulose from
Acetobacter resembles the crystalline structure and microfibrillar width of cellulose from plants and algae
(HOTCHKISS and BROWN, 1989; Ross et al., 1991). It was
characterized as highly crystalline, chemically pure,
metabolically inert, physically robust, highly absorbent,
and with less contaminating sugar and protein polymers
(HESTRIN and SCHRAMM, 1954; WHITE and BROWN,
1989; YAMANAKA et al., 1989; CANNON and ANDERSON,
1 To whom correspondence should be addressed. E-mail: l.Couperwhite@unsw.edu.au
600
E. B. BERNARDO et al.
601
Results
Morphological characterization and identification to
the generic level
A total of 38 cellulose producing isolates were isolated, purified and characterized. Of the 38 characterized
isolates, two strains designated as lTDI 2.1 (thick cellulose pellicle-producing strain) and PA 2.2 (thin cellulose
pellicle-producing strain) were selected for more detailed
characterization and identification. Formation of "nata"
cellulosic pellicle was observed in all 38 isolates. It is the
Fig. 1. Two types of cellulose pellicle floating on a clear broth medium: a) very thick, tough pellicle produced by strain ITDI 2.1, and
b) thin, soft pellicle produced by strain PA 2.2. The cells were incubated for 7 days at 30C in static condition. Arrows pointing to
the pellicles.
602
E. B. BERNARDO et al.
Source
ITDI 2.1
UPCC3
IN 103, IN 102
mother liquor/derivative of
pure culture
Mother liquor
CB 1.1,2.1,3.1
Mother liquor
Mother liquor
Mother liquor
Table 2. Groupings of the 38 nata-producing isolates based on colony and pellicle types.
Pellicle type
Colony type
Isolate codes
lTD! 2.1
B
UPCC3, PB7, LB 14a, BA 2 .3, NY 1.1, NY 2.1
Type A - thick, smooth and tough; Type B - moderately thick, smooth, firm to tough; Type C - thin, soft net-like appearance; Type 1
- smooth, circular, convex, shiny, tough, whitish to off-white; Type 2 - smooth, circular, convex, shiny, soft to firm, cream; Type 3 smooth, irregular, shiny, soft to firm, cream, release sticky exudates; Type 4 - rough, circular, shiny to dry, soft to firm, cream; Type
5 - rough/smooth, circular, shiny to dry, soft to firm, brownish.
603
Fig. 2. Two types of colony morphology: a) smooth colony type 1 by strain lTD! 2.1, and b) rough colony type 5 by strain PA 2.2.
Cells were streaked on PYSS plates and incubated at 30 DC for 7 days.
II OJ
B08
A TJlmu\
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53582
LB 06
LB 15a
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B
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LBp 1
LBI4
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BT 81
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23768
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LBp21
BT 93
-1L.._ _ _ _ __
102 5
I: cnlt
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10% Sucrose
7-S
Average (g 1- 1)
7% Sucrose
7-S
Average (g 1- 1)
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S.1S0.25
6.93 0.69
PA2.2
0.472
0.79
0.01
0.05
1.14
0.09
604
E. B. BERNARDO et al.
Table 4. Phenotypic characteristics of the two wild type Acetobacter strains at the species level.
Differentiating features
Formation of
Water-soluble brown pigment on GYC++
medium
Gluconic acid
Dhiydroxyacetone from glycerol
Growth on carbon sources:
Ethanol
Methanol
Sodium acetate
Growth on L-amino acids in the presence
of mannitol as carbon source:
L-asparagine
L-glutamine
Growth in 30% D-glucose
Growth in 10% ethanol
Catalase
Ubiquinone type" "
G+C Content (mol%)**
16S rDNA sequence
lTDI 2.1
PA2.2
ATCC
53582
A. xylinus"
A. pasteurianus':
A. hansenii'
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Discussion
It has always been speculated that the starter culture
used in producing cellulose nata pellicle is composed of a
heterogeneous population of microorganisms primarily
of different Acetobacter strains. But, there has not been
enough data to support this claim. Variations and differences exhibited by the 38 isolates in this study, based on
pellicle and colony types, indicated that there was heterogeneity in starter cultures used in nata de coco production. This may account for the observed batch to batch
differences in the type and volume of nata pellicle produced by the commercial producers. The two selected
strains, lTD I 2.1 and PA 2.2, explicitly demonstrated
these differences. Thick pellicle-forming strain lTD! 2.1,
showed a colony morphology distinct from the thin pellicle-forming strain PA 2.2. Colony type 1 morphology,
demonstrated by the thick cellulose pellicle-forming
strain, and the rest of the smooth colony forming isolates, is very different from a typical cellulose colony previously described. A typical colony produced by a cellulose synthesizing Acetobacter strain is described as initially smooth and spheroid and becoming rough, crinkled and flattened after 8 days incubation (DE LEY et aI.,
1984). In contrast, the colony produced by strain PA 2.2,
designated as colony type 5 and all the rest of rough
colony types in this study, conforms to the standard description of colony morphology for cellulose-producing
organisms except that, typically, these colonies were
raised not flattened after prolonged incubation. These results agreed with earlier works published on nata pelli-
606
E.
B. BERNARDO
et al.
under A. europaeus because ITDI 2.1 does not have a requirement for acetic acid for growth, whereas A. europaeus has an absolute requirement for acetic acid in
the growth medium (SIEVERS and TEUBER, 1995). On the
other hand, strain PA 2.2 having 98.2 % sequence similarity to A. hansenii, and having met the minimal phenotypic and biochemical requirement of this type species, is
proposed to be under this species designation. The derived percent similarities of the two strains ITD! 2.1
(98.7%) and PA 2.2 (98.2%), to two different type
species A. xylinus and A. hansen ii, respectively, strongly
supported the earlier assumption that these isolates were
distinctly different and may represent a novel and previously undescribed species of cellulose-synthesizing Acetobaeter. Speciation with Acetobacter, based on 16S
rDNA sequence similarities, has been warranted with as
little as 0.3 % sequence dissimilarity.
It was necessary to elucidate the species identification
of the two selected strains in order to establish which of
the three cellulose-producing species: A. xylinus, A. pasteurianus and A. hansenii they may belong. In the past,
there were significant changes in the nomenclature and
identification of cellulose producing Aeetobacter species
(DE LEY and FRATEUR, 1974; GOSSELE, 1983; YAMADA,
1983; DE LEY et a!., 1984). In the case of the nata-forming organism, the first correct identity, based on morphological and biochemical characteristics, was established
as belonging to Acetobacter xylinus subsp. Xylinus sensu
DE LEY and FRATEUR (LAPUZ et a!., 1967; GALLARDO-DE
JESUS et a!., 1973). Another nata bacterium obtained
from the Philippines was identified as Acetobaeter hansenii (GOSSELE and SWINGS, 1985) based on numerical
analysis of 177 phenotypic features (GOSSELE et a!.,
1983). This nata bacterium differed in a few characteristics from strain PA 2.2. It does not produce ketogluconic
acids from D-glucose and it has strong ketogenesis (dihydroxyacetone formation) towards polyalcohols whereas
the opposite in both tests were manifested by strain PA
2.2 (Table 4).
The reported cellulose dry weights for the two strains
were obtained from sucrose and fructose as carbon
sources where they grow favorably. Strain ITDI 2.1 can
also produce cellulose balls in shake culture condition
similar to A. xylinus subsp. sucrofermentans. In preliminary tests, it has been observed to produce an average
un optimized production yield of 2.50 and 3.70 g 1-1 in
sucrose and fructose, respectively, as compared to 4.4 g
1-1 optimum production yield of A. xylinus subsp. suerofermentans from Corn Steep liquor-Fructose medium
(TOYOSAKI et a!', 1995). Phylogenetic ally, strain lTD! 2.1
cannot be compared with A. xylinus subsp. sucrofermentans since there was no published 16S rRNA sequence
data for this organism. However, a few phenotypic differences were seen. ITDI 2.1 oxidizes fructose but not
lactose, produces abundant gluconic acid from glucose,
cannot grow in 10% ethanol but can grow minimally in
lactose, and shows growth in L-asparagine and L-glutamine in the presence of mannitol as the carbon source.
A. xylinus subsp. sucrofermentans showed the opposite
traits in all cases.
References
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and P. PIERTA) Rockville, Maryland 1992.
BETP: Nata de coco Exporters and Suppliers' (1993 to 1997)
Statistics. Manila, Philippines, Philippine Bureau of Export
and Trade Promotion (1997).
BIOLOG: Biolog Instruction Manual. Hayward, CA: Biolog Inc.
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BROWN, M. ].: Bacterial Cellulose, pp. 145-151. In: Cellulose:
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A. SKINNER, and D . LOVELOCK) London, Academic Press
1979.
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Manual of Determinative Bacteriology (R. E. BUCHANAN and
N. E. GIBBONS), 8th Edition, Baltimore, USA, The Williams
and Wilkins Co. 1974.
DE LEY, ]., GILLIS, M. and SWINGS, ].: Family VI. Acetobacteriacaea ., pp. 267-278. In: Bergey's Manual of Systematics Bacteriology., Vol. 1 (N. R. KREIG, and j. G. HOLT) Baltimore,
Williams and Wilkins, Co. 1984.
607
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E. B. BERNARDO et al.
Corresponding author:
1. COUPERWHITE, School of Microbiology and Immunology, University of New South Wales, Sydney, 2052 NSW, Australia
e-mail: LCouperwhite@unsw.edu.au