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Applied and Environmental Microbiology, December 1999, p. 5265-5271, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Biosynthesis of Novel Exopolymers by
Aureobasidium pullulans
Jin W.
Lee,1
Walter G.
Yeomans,2
Alfred L.
Allen,2
Fang
Deng,3
Richard A.
Gross,4,* and
David L.
Kaplan5,*
Dong-A University, Hadan 2-dong, Sha-gu,
Pusan, 604-714, Korea1; Biotechnology
Division, U. S. Army Natick Research, Development and Engineering
Center, Natick, Massachusetts 017602;
Department of Chemistry, University of
Massachusetts-Lowell, Lowell, Massachusetts
018543; Polymer Research Institute,
Polytechnic University, Brooklyn, New York
112014; and Biotechnology Center,
Department of Chemical Engineering, Tufts University, Medford,
Massachusetts 021555
Received 4 January 1999/Accepted 12 September 1999
 |
ABSTRACT |
Aureobasidium pullulans ATCC 42023 was cultured under
aerobic conditions with glucose, mannose, and glucose analogs as energy sources. The exopolymer extracts produced under these conditions were
composed of glucose and mannose. The molar ratio of glucose to mannose
in the exopolymer extract and the molecular weight of the exopolymer
varied depending on the energy source and culture time. The glucose
content of exopolymer extracts formed with glucose and mannose as the
carbon sources was between 91 and 87%. The molecular weight decreased
from 3.5 × 106 to 2.12 × 106 to
0.85 × 106 to 0.77 × 106 with
culture time. As the culture time increased, the glucose content of the
exopolymer extract formed with glucosamine decreased from 55 ± 3 to 29 ± 2 mol%, and the molecular weight increased from
2.73 × 106 to 4.86 × 106. There was
no evidence that glucosamine was directly incorporated into
exopolymers. The molar ratios of glucose to mannose in exopolymer extracts ranged from 87 ± 3:13 ± 3 to 28 ± 2:72 ± 2 and were affected by the energy source added. On the basis of the
results of an enzyme hydrolysis analysis of the exopolymer extracts and
the compositional changes observed, mannose (a repeating unit) was substituted for glucose, which gave rise to a new family of exopolymer analogs.
| |
INTRODUCTION |
Pullulan is one of the few neutral
water-soluble microbial polysaccharides that can be produced in large
quantities by fermentation (31, 38). Pullulan is an
extracellular, unbranched homopolysaccharide which consists of
maltotriose and maltotetraose units with both 
-(1
6) and
-(14) linkages (3, 6, 9, 38). The regular alternation
of -1,4 and -1,6 bonds results in two distinctive properties,
structural flexibility and enhanced solubility (22). These
properties suggest that pullulan may be used for both medical and
industrial purposes (23). Pullulan produces high-viscosity solutions at relatively low concentrations and can be utilized to form
oxygen-impermeable films, thickening or extending agents, or adhesives
(28). Films formed from pullulan are suitable for coating
foods and pharmaceuticals, especially when exclusion of oxygen is
desirable (46).
Pullulan biosynthesis is accomplished through mediation of sugar
nucleotide-lipid carrier intermediates associated with the cell
membrane fraction (8, 38). Some important parameters that
control the production of pullulan are temperature (28), the
initial pH of the medium (15, 18, 30), the oxygen supply (32, 41), the nitrogen concentration (1, 35), and
the carbon source (2). The molecular weight of pullulan
varies depending on the culture conditions and strain (31, 36,
44).
In our attempts to modify the native structure of polysaccharides, we
recently examined the effects of glucose analogs when they were used as
energy sources for the production of exopolymers (24-26).
In some cases, novel exopolymers were formed, or the glucose analogs
affected the molecular weight or composition of the native exopolymers
synthesized. In this paper we describe the influence of glucose
analogs, such as 3-O-methyl-D-glucose
(3-O-methylglucose), 2-amino-2-deoxy-D-glucose
(glucosamine), and 2-acetamino-2-deoxy-D-glucose (N-acetylglucosamine) on the yield and composition of
exopolymers synthesized by Aureobasidium pullulans. We also
explored the possibility that these glucose analogs might be directly
polymerized by A. pullulans. Our goals were to devise
biosynthesis strategies which modulate the composition and structural
features of exopolymers and to gain insight into the metabolic
flexibility of the biosynthesis and secretion pathways.
| |
MATERIALS AND METHODS |
Bacterial strain.
A. pullulans ATCC 42023 (47) was obtained from the American Type Culture Collection
and was transferred monthly to fresh nutrient agar medium. The medium
used for cell growth and exopolymer production contained (per liter)
5.0 g of K2HPO4, 1.0 g of NaCl, 0.2 g of MgSO4 · 7H2O, 0.6 g
of (NH4)2SO4, 2.5 g of yeast
extract, and 20 g of glucose (39). The pH of medium was
adjusted to 6.5 to 6.7 before sterilization. Each carbohydrate source
was autoclaved separately for 20 min at 121°C and was added to the
medium under aseptic conditions.
Production of exopolymer.
Starter cultures were prepared by
transferring cells from agar slants to 50-ml portions of medium
containing 2% (wt/vol) glucose in 250-ml Erlenmeyer flasks. The
resulting cultures were incubated for 2 days at 30°C and 180 rpm.
Each starter culture was used as an inoculum (3%, vol/vol) for 150 ml
of medium supplemented with an energy source (2%, wt/vol) in a 500-ml
Erlenmeyer flask. The cultures were incubated for 5 days under the same
conditions used to prepare the starter cultures. The energy sources,
including glucose (>99.5% pure) and glucose-related sugars (>99.0%
pure), such as 3-O-methylglucose, glucosamine, and
N-acetylglucosamine, were purchased from Sigma Chemical Co.
(St. Louis, Mo.). Samples were periodically withdrawn from the cultures
to examine cell growth and exopolymer production.
Purification of exopolymer.
Cell broth was centrifuged at
15,000 × g for 20 min at 4°C to remove the cells. To
determine biomass, the cells were washed with distilled water and dried
at 100 to 105°C until the weight was constant. Supernatant fluids
were mixed with 2 volumes of 95% ethanol and incubated at 4°C for
24 h to precipitate the crude products, which were separated by
centrifugation at 15,000 × g for 30 min. The
precipitated material was repeatedly washed with acetone and ether,
dissolved in deionized water, and dialyzed against deionized water by
using dialysis tubing with a molecular weight cutoff of 12,000 to
14,000. After dialysis for 2 to 3 days with four or five changes of
deionized water, the solution was lyophilized, and the exopolymer yield
was determined by weighing.
Treatment with pullulanase and chemical analysis.
Exopolymers were assayed for sensitivity to pullulanase (21,
43). Exopolymers were suspended at a concentration of 1 mg/ml (0.1%, wt/vol) in 50 mM sodium acetate buffer (pH 5.0). Pullulanase from Klebsiella pneumoniae (Sigma Chemical Co.) was added to
a concentration of 0.1 U/ml. After mixing, the treated samples were incubated for 42 h at 25°C. Authentic pullulan (Sigma Chemical Co.) was digested as a control, and data are reported below as percentages of reducing sugars relative to complete hydrolysis to
maltotriose units.
Total-sugar and reducing-sugar contents were determined by the phenol
sulfuric acid method (12) and the dinitrosalicylic acid
(DNS) method, respectively (29, 33). DNS reagent was prepared by first dissolving 7.46 g of 3,5-DNS and 13.98 g of NaOH
pellets in 1 liter of deionized water. Then 216.1 g of Rochelle Salt
(potassium sodium tartarate tetrahydrate), 5.38 ml of saturated phenol,
and 5.85 g of sodium metabisulfite were added, and the reagent was
aged for 2 weeks. DNS reagent was added to the same volume of an
enzyme-substrate solution, and the preparation was placed in a boiling
water bath for 15 min. After the preparation cooled to room
temperature, the concentration of reducing sugars was determined
spectrophotometrically at 550 nm with a spectrophotometer (Beckman
Instrument Co.). The calibration curve used for reducing-sugar determinations was generated by using maltotriose (Sigma Chemical Co.).
Composition analysis by GC.
Gas chromatography (GC) and
GC-mass spectrometry (MS) were used to determine the carbohydrate
composition after methanolysis and trimethylsilyation of the product
(10). Samples were prepared for GC-MS analyses as described
elsewhere (10, 24). GC-MS analyses were performed with a
Hewlett-Packard gas chromatograph (model 5890 series II) equipped with
an Hewlett-Packard model 7673 injector and coupled to a mass selective
detector (Hewlett-Packard model 5971 series). The capillary column used
was a cross-linked 5% phenylmethyl silicone fused-silica column (HP
Ultra MS 5; 30 m by 0.25 mm; film thickness, 0.33 µm). Dry
oxygen-free helium (flow rate, 0.8 ml/min) was used as the carrier gas,
and the column temperature was programmed so that it was 140°C for 2 min and then increased at a rate of 8°C per min to 260°C.
One-microliter samples were injected, and the injector was purged for
0.6 min after injection. m-Inositol was used as the internal standard.
Determination of molecular weight by GPC.
The number average
molecular weight (Mn) (average molecular weight
divided by the number of molecules) and the weight average molecular
weight (Mw) (average molecular weight divided by
the weight of each polymer chain), as well as the polydispersity
(Mw/Mn) (the breadth of
the molecular weight distribution) of pullulan samples, were determined
by gel permeation chromatography (GPC) by using a Waters model 600E
system controller equipped with Shodex KB800 series columns (two KB80M
columns and one KB805 column) and a model 410 refractive index
detector. All data processing was carried out by using Millennium
version 2.15 software. Pullulan standards (Polysciences) with narrow
polydispersity and with molecular weights ranging from 5.80 × 103 to 8.53 × 105 were used to construct
a calibration curve. Deionized water containing 0.05% (wt/vol) sodium
azide was used as the mobile phase at a flow rate of 1.0 ml/min. The
sample concentration and injection volume were 5.0 mg/ml and 100 µl,
respectively. All of the sample solutions were filtered through
0.45-µm-pore-size filters before injection. For the samples which
produced multimodal distribution GPC chromatograms, a curve fit
software program (Peakfit, version 4.0, for Win32; Jandel Scientific
Software Inc.) was used to analyze the GPC chromatograms
(r2 > 0.950). The GPC chromatograms were
estimated, relative to the pullulan standards, by assuming Bernoullian
shape curves and using three molecular weight range components for each chromatogram.
| |
RESULTS |
Monomeric composition of exopolymers.
The GC chromatograms of
the exopolymers purified from the culture grown with 2% (wt/vol)
glucose showed that the major component was glucose, as identified by
the peak retention times for the and 
anomers (9.34 and 9.82 min, respectively) and the area ratios of these anomers to a pure
standard (10.5 to 1.0) (10). The exopolymer synthesized with
2% (wt/vol) glucosamine consisted of glucose and mannose, as
determined by GC. The identities of the glucose and mannose in the
GC-MS chromatograms of exopolymers were confirmed by using reference
spectra in the GC-MS data bank (data not shown).
Production of exopolymer with glucose analogs.
Cell growth and
exopolymer production were greater with glucose than with any of the
other energy sources used under the conditions used (Table
1). 3-O-Methylglucose and
2-deoxyglucose were not utilized effectively by A. pullulans
ATCC 42023. Cell growth with glucosamine was similar to cell growth
with N-acetylglucosamine. The exopolymers synthesized with
glucose, 3-O-methylglucose, glucosamine, N-acetylglucosamine, 2-deoxyglucose, and mannose were
analyzed by GC. All of the exopolymers produced similar GC peaks which corresponded to only glucose and mannose. Based on the relative peak
areas and the response factors for corresponding standard sugars, the
relative glucose and mannose contents of the exopolymers were
calculated (Table 1). The glucose and mannose contents of the
exopolymer synthesized with 2% (wt/vol) glucose were 87 ± 3 and
13 ± 3 mol%, respectively, whereas the glucose and mannose contents of the exopolymer synthesized with 2% (wt/vol) glucosamine were 30 ± 7 and 70 ± 7 mol%, respectively. The ratio of
glucose to mannose in the exopolymer synthesized with 2% (wt/vol)
mannose was similar to the ratio of glucose to mannose in the
exopolymer synthesized with 2% (wt/vol) glucose (Table 1).
Production of exopolymers as a function of cultivation time.
Cell growth and production of exopolymers when glucose, mannose, and
glucosamine were used as energy sources were compared as a function of
time. On the basis of cell dry weight, the growth of A. pullulans cells depended on the energy source (Fig.
1A). The highest exopolymer yields with
glucose and mannose were obtained after 3 and 2 days of growth,
respectively (Fig. 1B). The production of exopolymers in the presence
of glucose, mannose, and glucosamine paralleled cell growth. The molar
ratio of glucose to mannose in exopolymers synthesized with glucose and
mannose remained almost constant as a function of culture time (about
90:10) (Fig. 1C). The molar ratio of glucose to mannose in the
exopolymer synthesized with glucosamine decreased with culture time.
The initial ratio of glucose to mannose was 55 ± 3:45 ± 3;
after 5 days the ratio was 29 ± 2:71 ± 2.
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FIG. 1.
Production of exopolymers with glucose ( ), mannose
( ), and glucosamine ( ) as a function of time of cultivation of
A. pullulans ATCC 42023. (a) Cell growth. (b) Exopolymer
production. (C) Molar ratio of glucose to mannose in exopolymers.
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|
GPC chromatograms of exopolymers purified from 5-day cultures in which
glucose, mannose, and glucosamine were the energy sources
showed that
the molecular weight of each exopolymer was heterogeneous
(Fig.
2). On the basis of molecular weight, the
exopolymers could
be divided into high-molecular-weight (molecular
weight, more
than 1.0 × 10
6), medium-molecular-weight
(1.0 × 10
6 to 5.0 × 10
4), and
low-molecular-weight (less than 5.0 × 10
4) fractions.
The relative amounts of the high- and medium-molecular-weight
fractions
of exopolymer synthesized with glucose decreased as
the amount of the
low-molecular-weight fraction of exopolymer
increased (Fig.
3). As a function of culture time, the
low-molecular-weight
fractions of the exopolymers synthesized with
glucose and mannose
dominated, whereas the high-molecular-weight
fractions of the
exopolymer synthesized with glucosamine accounted for
90% of total
exopolymer (Fig.
4). Thus,
the major fraction of the exopolymers
synthesized with glucose and
mannose was the low-molecular-weight
fraction, but the major fraction
of the exopolymer synthesized
with glucosamine was the
high-molecular-weight fraction.
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FIG. 2.
GPC chromatograms of exopolymers purified from 5-day
cultures grown on glucose (A), mannose (B), and glucosamine (C).
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FIG. 3.
Molecular weight fractional patterns as a function of
culture time. (A) Glucose. (B) Mannose. (C) Glucosamine. Symbols: ,
high-molecular-weight fraction; , medium-molecular-weight fraction;
 , low-molecular-weight fraction.
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FIG. 4.
Patterns of molecular fraction areas as a function of
culture time when glucose (A), mannose (B), and glucosamine (C) were
the carbon sources. Black bars, high-molecular-weight fraction; gray
bars, medium-molecular-weight fraction; white bars,
low-molecular-weight fraction. MW, molecular weight.
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|
Production of exopolymers with mixed carbon sources.
The molar
ratios of glucose to mannose in the exopolymers synthesized after
incubation for 5 days with 2% (wt/vol) glucose and 2% (wt/vol)
glucosamine as the sole energy sources were 87 ± 3:13 ± 3 and 28 ± 2:72 ± 2, respectively (Table
2). The molar ratios of glucose to
mannose in exopolymers synthesized with glucose and glucosamine
together were between the molar ratios in the exopolymers synthesized
with the individual energy sources. The cell growth and exopolymer
yield obtained with glucose plus glucosamine as the carbon source
exhibited responses which reflected the relative ratios of glucose and
mannose in the polymer. The exopolymer synthesized with a higher
relative concentration of glucosamine in the mixed energy source had a
higher mannose content.
Pullulan and exopolymers synthesized with glucose and mixtures of
glucose and glucosamine contained increased concentrations
of reducing
sugars after treatment with pullulanase (Fig.
5).
After a 12-h treatment, the reducing
sugars from pullulan were
86 ± 5.1% maltotriose equivalents. The
reducing-sugar contents
of exopolymers with molar ratios of glucose to
mannose of 89:11
(exopolymer 1), 79:21 (exopolymer 2), and 55:45
(exopolymer 3)
were 93 ± 9.2, 73 ± 4.3, and 31 ± 5.0%, respectively.
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FIG. 5.
Treatment of pullulan ( ), exopolymers synthesized
with glucose (molar ratio of glucose to mannose, 89:11) (),
exopolymers synthesized with glucose and glucosamine (molar ratio of
glucose to mannose, 79:21) (), and exopolymers synthesized with
glucosamine (molar ratio of glucose to mannose, 55:45) () with
pullulanase.
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| |
DISCUSSION |
Based on the data reported here, exopolymers synthesized by
A. pullulans may consist of a family of glucose-mannose
copolymers or may involve modulation of the composition of pullulan
itself. It is important to note that in previous studies of pullulan
the workers usually did not clarify the level of mannose in the
polymer(s) and there was no attempt to separate different polymers, if
they were present. The yield of exopolymers produced by A. pullulans and the relative amount of pullulan (usually defined as
the ethanol precipitate) have been reported to vary with the culture
conditions and strain (4, 21, 30, 42, 43). Depending on the
strain and carbon source, the pullulan content of the exopolymer
produced by A. pullulans has varied from 95 to 73%
(30), from 76 to 51% (43), or from 100 to 45%
(43). Exopolymers purified from the fermentation broth media
of A. pullulans cultures grown with various agroindustrial
wastes were heterogeneous with respect to monomeric composition and
molecular weight (16). For example, the glucose contents of
the exopolymers synthesized with olive oil waste effluents and molasses
as the carbon sources were about 33 and 25%, respectively, while all
of the monomeric components of exopolymers synthesized with starch
waste and grape skin pulp extract were glucose. The exopolymer
synthesized with carob pod extract as the energy source contained only
glucose, and the glucosidic linkages were primarily -(14) (68%)
and -(16) (31%) linkages (34).
The pullulan content of exopolymers synthesized with glucose as the
energy source decreased with culture time (4). The major
component of the extracellular polysaccharides produced by A. pullulan with glucose as the energy source was glucose, and the
minor components were mannose and galactose (47). In the
present study, the exopolymers produced by A. pullulan ATCC 42023 in the presence of glucose and its analogs consisted of glucose
and mannose. The molar ratio of glucose to mannose in the exopolymers
varied from 90:10 to 30:70 depending on the energy source and culture
time. The energy source was presumably responsible for determining the
repeat unit composition of the exopolymer produced by A. pullulan.
Beijerinckia indica utilized glucose, as well as glucose
analogs, such as 3-O-methylglucose, glucosamine,
N-acetylglucosamine, and 2-deoxyglucose, for growth and
produced polysaccharide 7. However, there was no evidence that these
sugars were directly incorporated into exopolymers, and there was no
change in the repeat unit composition of polysaccharide 7 (25). Two of the glucose analogs,
3-O-methylglucose and N-acetylglucosamine, were directly incorporated into exopolysaccharides produced by
Agrobacterium sp. (26). When
3-O-methylglucose was used, 8 to 12 mol% of the curdlan
repeats were 3-O-methylglucose based on GC and
1H nuclear magnetic resonance spectrometry data. When
glucose analogs were used as energy sources, they did not support
Zoogloea ramigera cell growth. However, when mixtures of
glucose and these sugars were used, as cosubstrates, they supported
cell growth and resulted in a significant change in the internal ratio
of components (26). The use of glucose analogs as
cosubstrates during Z. ramigera cultivation resulted in
dramatic changes in sugar metabolism due to the competition of these
compounds with glucose (11, 40) and in alterations in the
gene expression involved in the biosynthetic pathway of zooglan
(13). When glucose analogs are used as energy sources with
A. pullulan, they appear to modulate sugar metabolism, which
results in changes in the composition of exopolysaccharides.
The average molecular weight of pullulan ranges from 1.5 × 104 to 1.0 × 107 depending on the culture
conditions and strain used (31, 36, 44). The molecular
weight of pullulan decreased late in the stationary growth phase due to
the presence of the less frequent amylase-sensitive maltotetraose sites
among the predominantly maltotriose units in pullulan and due to
-amylase secreted into the medium (5, 20, 31). New
strains were isolated, and the pH used for cultivation was optimized to
produce higher-molecular-weight pullulan (27, 31).
Exopolymers produced by A. pullulans with different
substrates had different molecular weights and repeat unit compositions (16). The molecular weights of exopolymers synthesized with agricultural wastes were higher than the molecular weights of exopolymers synthesized with glucose as the energy source. In this
study, exopolymers synthesized with glucose as well as mannose had a
monomeric composition similar to that of pullulan. The molecular weights of these exopolymers decreased with culture time, while the
molecular weights of exopolymers synthesized with glucose analogs,
which had high mannose contents, did not decrease with culture time.
Apparently, the -amylase or the pullulan-degrading enzymes produced
by the cells (20) cannot hydrolyze the exopolymers synthesized with glucose analogs due to the change in mannose content.
Pullulanase is one of the starch-debranching enzymes that specifically
attacks the branch points of amylopectin, hydrolyzing -(1,6)glucosidic linkages to produce maltotriose (17, 37, 45). The increase in reducing sugar content after pullulanase treatment of pullulan and the exopolymers synthesized with glucose and
glucose analogs indicated that the exopolymers have -(1,6)glucosidic linkages like those in pullulan. These data suggest that the
exopolymers have triose units, like pullulan. We confirmed that the
exopolymers purified from the cultures grown on glucose had a structure
identical to that of pullulan based on 13C nuclear magnetic
resonance chromatograms (data not shown). The exopolymers with
increased mannose contents exhibited resistance to hydrolysis by
-amylase.
The biosynthetic pathway for the exopolymers produced by A. pullulan has not been well established (8, 38). There
are two possibilities for incorporation of mannose into the exopolymers synthesized by A. pullulans. One option involves an
epimerase that converts glucose to mannose after polymerization.
Epimerization by C-5-mannuronan epimerase has been found in
Pseudomonas aeruginosa (14) but has not been
reported in A. pullulans. The monomeric ratio of mannuronate
to guluronate in exopolymer synthesized by P. aeruginosa
changed depending on the activity of C-5 epimerase, which was affected
by the concentration of salts in culture medium (19). The
other possibility for incorporation of mannose into the exopolymer is
direct incorporation. The only monomeric components of exopolymers
synthesized with energy sources including glucose analogs in this
experiment were glucose and mannose, but the ratios were different
depending on the growth conditions. Direct incorporation of glucose and
mannose into exopolymers may be affected by the physiological
conditions, including energy source and culture time, that were changed
in this study.
In this study, exopolymers with various molar ratios of glucose to
mannose, which ranged from 87:13 to 28:72, were synthesized during
growth on glucose and glucose analogs. The ratio of glucose to mannose
in exopolymers can be controlled by the ratio of mixed carbon sources.
Therefore, it is possible to produce exopolymers with a more defined
ratio of glucose to mannose. These exopolymers have higher molecular
weights and narrower polydispersity than pullulan. Further work to
characterize the structural and functional properties of these
exopolymers with various molar ratios of glucose to mannose produced by
A. pullulans is needed.
| |
FOOTNOTES |
*
Corresponding author. Mailing address for Richard A. Gross: Polymer Research Institute, Polytechnic University, Brooklyn, NY
11201. Phone: (718) 260-3024. Fax: (718) 875-9646. E-mail: rgross{at}pdy.edu. Mailing address for David L. Kaplan: Biotechnology Center, Department of Chemical Engineering, Tufts University, Medford,
MA 02155. Phone: (617) 627-3251. Fax: (617) 627-3991. E-mail:
dkaplan1{at}tufts.edu.
| |
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Applied and Environmental Microbiology, December 1999, p. 5265-5271, Vol. 65, No. 12
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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