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Applied and Environmental Microbiology, January 2000, p. 49-53, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Establishment of a Polychlorinated Biphenyl-Dechlorinating
Microbial Consortium, Specific for Doubly Flanked Chlorines, in a
Defined, Sediment-Free Medium
Qingzhong
Wu,1
Kevin R.
Sowers,2 and
Harold D.
May1,*
Department of Microbiology and Immunology,
Medical University of South Carolina, Charleston, South
Carolina,1 and Center of Marine
Biotechnology, University of Maryland Biotechnology Institute,
Baltimore, Maryland2
Received 28 June 1999/Accepted 22 October 1999
 |
ABSTRACT |
Estuarine sediment from Charleston Harbor, South Carolina, was used
as inoculum for the development of an anaerobic enrichment culture that
specifically dechlorinates doubly flanked chlorines (i.e., chlorines
bound to carbon that are flanked on both sides by other chlorine-carbon
bonds) of polychlorinated biphenyls (PCBs). Dechlorination was
restricted to the para chlorine in cultures enriched with
10 mM fumarate, 50 ppm (173 µM) 2,3,4,5-tetrachlorobiphenyl, and no
sediment. Initially the rate of dechlorination decreased upon the
removal of sediment from the medium. However, the dechlorinating activity was sustainable, and following sequential transfer in a
defined, sediment-free estuarine medium, the activity increased to
levels near that observed with sediment. The culture was
nonmethanogenic, and molybdate, ampicillin, chloramphenicol, neomycin,
and streptomycin inhibited dechlorination activity;
bromoethanesulfonate and vancomycin did not. Addition of 17 PCB
congeners indicated that the culture specifically removes double
flanked chlorines, preferably in the para position, and
does not attack ortho chlorines. This is the first
microbial consortium shown to para or meta
dechlorinate a PCB congener in a defined sediment-free medium. It is
the second PCB-dechlorinating enrichment culture to be sustained in the
absence of sediment, but its dechlorinating capabilities are entirely different from those of the other sediment-free PCB-dechlorinating culture, an ortho-dechlorinating consortium, and do not
match any previously published Aroclor-dechlorinating patterns.
| |
INTRODUCTION |
Microbial reductive dechlorination
of polychlorinated biphenyls (PCBs) under anaerobic conditions has been
shown to occur in freshwater, brackish, and marine sediments (reviewed
in reference 2). The isolation of PCB-dechlorinating
microorganisms is necessary to better understand the physiological
mechanisms and catalysis of microbial PCB dechlorination. However, all
reported attempts to isolate microorganisms that catalyze these
reactions have been unsuccessful. Most of the PCB-dechlorinating
enrichment cultures reported are derived from PCB-contaminated
freshwater sediment (2, 4, 8, 17, 20, 22). The
dechlorinating activities of these cultures are directed at
meta and/or para chlorines and have not been
demonstrated in the absence of soil or sediment. The transformation of
biogenic halogenated organic compounds in estuarine and marine
environments suggests that haloaromatic dechlorinators, including PCB
dechlorinators, are present in these habitats. Recently it has been
reported that microorganisms derived from estuarine sediment from
Baltimore Harbor (Baltimore, Md.) are able to dechlorinate single PCB
congeners (5) and Aroclor 1260 (19) by removal of
meta, para, and ortho chlorines. A
2,3,5,6-tetrachlorobiphenyl (2,3,5,6-CB),
ortho-dechlorinating culture has been enriched from these
sediments, and the culture has since been transferred and sustained in
a sediment-free medium (8).
Herein we report on the meta and para
dechlorination of 2,3,4,5-CB and freshly added Aroclor 1260 in
estuarine sediment from Charleston Harbor (Charleston, S.C.). A stable
PCB-dechlorinating consortium (2,3,4,5-CB enrichment culture) was
developed from that source of sediment. The culture attacks only doubly
flanked chlorines of PCBs, chlorines that are flanked on each side by another chlorine bound to a carbon atom, indicating a high degree of
specificity for PCB dechlorination.
| |
MATERIALS AND METHODS |
Sediment sample.
Sediment samples were collected with a
petite Ponar grab sampler at a subsurface depth of 4 m in the
Ashley River branch of Charleston Harbor (32°47.1'N, 79°57.5'W).
Sediments had a black coloration and gelatinous texture. The combined
contents of the sampler were transferred to 0.95-liter canning jars
(Ball Corporation, El Paso, Tex.). The jars were filled to the top and
immediately sealed with dome tops and ring seals to exclude air. The
samples were stored at ambient temperature in the dark prior to use.
Culture procedures.
An estuarine medium (E-Cl) was prepared
as described by Berkaw et al. (5), except that
Na2S · H2O was not added. The final pH
of the medium was 7.0. Charleston Harbor sediment, when used as a
medium component, was dried, ground, and autoclaved twice for 1 h
at 121°C on two consecutive days before use. The medium was
autoclaved at 121°C for 30 min. Estuarine sediment from Charleston Harbor (2 ml) was inoculated into 8 ml of E-Cl medium. Aroclor 1260, in
10 µl of acetone, was added to a final concentration of 800 µg/g
(dry weight) of sediment. The congeners 2,3,4,5-CB and 2,3,5,6-CB, in
10 µl of acetone, were added to a final concentration of 173 µM (50 ppm) or to 350 µM when they were added with the Aroclor. Cultures
containing Aroclor were prepared in triplicate, and cultures maintained
with single congeners only were prepared in duplicate. The
Aroclor-containing cultures were supplemented monthly with 2.5 mM
concentrations (each) of sodium acetate, propionate, and butyrate. All
cultures were incubated at 30°C in the dark.
Specificity test of PCB dechlorination.
E-Cl medium was
inoculated with the 2,3,4,5-CB enrichment culture (1%, vol/vol,
transfer from sediment or sediment-free culture). Each culture
contained 10 mM fumarate and one of following congeners (final
concentration, 100 µM): 3-CB, 4-CB, 2,3-CB, 2,4-CB, 2,5-CB, 3,4-CB,
3,5-CB, 2,3,4-CB, 2,3,5-CB, 2,3,6-CB, 2,4,6-CB, 3,4,5-CB, 2,3,4,5-CB,
2,3,4,6-CB, 2,3,5,6-CB, 2,3,4,5,6-CB, and 2,4,5-2,4,5-CB.
Analytical procedures.
Cell growth in sediment-free
enrichment cultures was monitored by measuring the change in optical
density at 600 nm with a Spectronic 20D spectrophotometer (Milton Roy,
Rochester, N.Y.). Methane concentrations were analyzed on a
Hewlett-Packard (Atlanta, Ga.) 5890 series gas chromatograph (GC)
equipped with a model RTX-624 capillary column {30 m by 0.53 mm
(inside diameter [i.d.]) by 0.3 µm; Restek Corp., Bellefonte,
Pa.} and a flame ionization detector. The temperatures of the oven,
injector, and detector were 80, 240, and 325°C, respectively.
PCBs were extracted from enrichment cultures with ethyl acetate
(high-performance liquid chromatography grade; Fisher Scientific, Pittsburgh, Pa.), and the solvent extracts were passed through a
Florisil-copper column (5). PCBs were analyzed with a
Hewlett-Packard 5890 series GC equipped with a model RTX-1 capillary
column (30 m by 0.25 mm [i.d.] by 0.25 µm; Restek Corp.) and an
Ni63 electron capture detector as described previously
(5). PCBs were identified by matching their GC retention
times with those of authentic standards (99% purity;
AccuStandard) and quantified with a 6- to 16-point calibration curve
for each congener (5). The congeners 2,4-CB and 2,5-CB or
2,3,5,6-CB and 2,3,4,6-CB could not be separated by this method and
so were reported together. Biphenyl was assayed on a Hewlett-Packard
6890 series GC equipped with a model HP-5MS capillary column (30 m by
0.25 mm [i.d.] by 0.25 µm; Hewlett-Packard) and a Hewlett-Packard
6890 series mass selective detector.
Analysis of Aroclor was done according to the method of Wu et al.
(
19). PCB extraction, preparation, and analysis by GC
and
electron capture detection were done in a manner similar to
that
described above. Aroclor 1260 and dechlorination products
were
identified by matching GC retention times with those of a
customized
PCB standard prepared by supplementing Aroclor 1260
with the
dechlorination products observed in Woods Pond (L. A.
Smullen,
K. A. DeWeerd, D. L. Bedard, W. A. Fessler, J. C. Carnahan,
and R. E. Wagner, p. 45-6,
in Twelfth
progress report of research
and development program for the destruction
of PCBs, General Electric
Co., Schenectady, N.Y., 1993) or with a
standard mixture composed
of 3-3-CB, 3-4-CB, 3,5-3-CB, 3,5-4-CB,
2,4-3,5-CB, and 2,5-3,5-CB.
Congener assignments were made according to
those reported by
Frame et al. (
9). Each congener in the
Aroclor mixture was
quantified by use of a piecewise-fit calibration
curve generated
from standards at 4- to 8-point calibration levels.
Congener and
homolog distribution for each sample were calculated and
reported
in units of moles percent, which were used to calculate the
chlorine/biphenyl
ratios. Congener distributions for each enrichment
culture with
Aroclor and 2,3,4,5-CB (or 2,3,5,6-CB) were calculated
after subtracting
the peaks corresponding to 2,3,4,5-CB (or 2,3,5,6-CB)
and their
potential dechlorination
products.
| |
RESULTS |
Dechlorination of Aroclor 1260 with Charleston Harbor
sediment.
Aroclor 1260 is primarily composed of hexa- and
heptachlorobiphenyls. A portion (25.3 ± 1.7% [mean ± standard deviation]) of the chlorines associated with Aroclor 1260 was
removed by reductive dechlorination following 6 months of incubation
with Charleston Harbor sediment (Fig. 1).
The major congeners in Aroclor 1260 include 2,3,4-2,4,5-CB,
2,4,5-2,4,5-CB, 2,3,6-2,4,5-CB, 2,3,4,5-2,3,6-CB, 2,3,4,5-2,4,5-CB, and 2,3,5,6-2,4,5-CB, while the most prevalent dechlorination products were 2,4-2,4-CB, 2,4-2,5-CB, 2,5-2,5-CB, 2,4-2,6-CB, and 2,5-2,6-CB. This activity was heavily dominated by
dechlorination of meta chlorines (60.1 ± 2.8%) with a
trace of para dechlorination (4.3 ± 0.4%). The
addition of congener 2,3,4,5-CB hastened the onset of
dechlorination of the Aroclor 1260 by at least 1 month. Total
dechlorination increased modestly to 29.8 ± 0.7% when 2,3,4,5-CB
was added. Most of this increase was due to enhanced para
dechlorination (11.2 ± 0.6%) since only a slight increase in
meta dechlorination was observed (66.0 ± 2.1%). The
onset of Aroclor dechlorination was also hastened by the addition
of congener 2,3,5,6-CB, and meta dechlorination was slightly enhanced, but this congener failed to stimulate
para dechlorination. No ortho dechlorination was
observed under any conditions with the Charleston sediment. These
results stand in contrast to those obtained with Baltimore Harbor
sediment (19), where more overall dechlorination occurred
due to ortho dechlorination.
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FIG. 1.
Dechlorination of Aroclor 1260 in Charleston Harbor
sediments incubated in E-Cl medium. Datum points are the means ± standard deviations of results from triplicate cultures.
Chlorine/biphenyl ratios are presented for ortho ( )-,
meta ( )-, and para ( )-positioned chlorines.
Shown are data from microcosms incubated with 800 µg of Aroclor 1260 per g (dry weight) of sediment (A), with Aroclor plus 350 µM
2,3,4,5-CB (B), and with Aroclor plus 350 µM 2,3,5,6-CB (C).
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|
Establishment of a para-dechlorinating culture.
The Aroclor data clearly established that meta- and
para-PCB dechlorination occurred with the Charleston Harbor
sediments and that 2,3,4,5-CB could further stimulate this activity. An enrichment series with 10% (vol/vol) Charleston Harbor sediment and
173 µM 2,3,4,5-CB was started in E-Cl medium. Initially, both meta and para dechlorination of 2,3,4,5-CB was
observed in these cultures. In the absence of a defined carbon source,
congener 2,3,4,5-CB was dechlorinated to 2,3,5-CB, 2,4,5-CB, and 2,4-CB or 2,5-CB (Fig. 2A). The culture clearly
favored a combination of meta and para
dechlorination, with >75% of the parent congener being converted to
2,4-CB or 2,5-CB and no appearance of ortho-dechlorination products. At this point, 10 µl of actively dechlorinating sediment slurries were transferred into 10 ml of E-Cl medium containing 5%
(wt/vol) sediment. After two sequential transfers, 2,3,5-CB became the
sole dechlorination product of 2,3,4,5-CB and the
meta-dechlorination activity was no longer observed (Fig.
2B). After four additional transfers, the amount of sediment added to
the medium was reduced to 0.1% (wt/vol) and the specificity of
dechlorination remained the same. When different carbon and energy
sources (acetate, butyrate, crotonate, formate, fumarate, lactate,
malate, propionate, pyruvate, and succinate) were added to the culture,
fumarate enhanced the rate of para dechlorination of
2,3,4,5-CB by an average of 114%. Henceforth, the medium was
supplemented with 10 mM fumarate as the sole carbon and energy source.
The dechlorination pattern and rate have remained relatively constant
under these conditions for more than 12 sequential transfers over the
course of 25 months.
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FIG. 2.
Dechlorination of 2,3,4,5-CB by a primary enrichment
culture with Charleston Harbor sediment (A) and by the second
sequential transfer (duplicate cultures) of the primary enrichment
culture (B). PCB congeners represented are 2,3,4,5-CB (), 2,3,5-CB
( ), 2,4,5-CB ( ), and 2,4- or 2,5-CB ( ).
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Following six sequential transfers (1%, vol/vol) into E-Cl medium with
0.1% sediment (dry weight), the active culture was
sequentially
transferred five times into E-Cl medium containing
10 mM fumarate
without added sediment. Compared with the dechlorination
activity in
the presence of sediment, the activity in the absence
of sediment was
slower (Fig.
3). However, after three
sequential
transfers, the rate and extent of dechlorination had
returned
to levels similar to those observed with cultures maintained
with
sediment (Fig.
4 and
5). No methane gas was detected in the
sediment-free
cultures. Following the growth of cultures, the results
from which
are depicted in Fig.
4, the
para-dechlorination
activity in sediment-free
cultures could be retained in dilutions up to
10
5 (vol/vol), indicating that the microbial catalyst(s)
accounted
for at least 10
5 cells per ml in the culture.
Figure
4 also shows that most of
the increase in optical density had
occurred before most of the
dechlorination had taken place. High
optical densities were not
achieved, but the data indicate that growth
did continue at a
slow pace while dechlorination continued.
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FIG. 3.
Numbers of chlorines per biphenyl for the first
generation of sediment-free 2,3,4,5-CB enrichment cultures ( ) and
the cultures with 0.1% sediment (). Datum points are the means of
results from duplicate cultures.
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FIG. 4.
Optical densities () and numbers of chlorines per
biphenyl () of the fourth sequential transfer of the
2,3,4,5-CB-dechlorinating consortium in the absence of sediment. Datum
points are the means of results from duplicate cultures.
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FIG. 5.
Dechlorination of 2,3,4,5-CB by the sediment-free
dechlorinating consortium with 2 mM BES (), 100 µg of vancomycin
per ml (), or no inhibitor (). Datum points are the means of
results from duplicate cultures.
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Specificity of PCB dechlorination.
The specificity of PCB
dechlorination of the 2,3,4,5-CB enrichment culture was investigated
after five sequential transfers of the culture in the absence of
sediment and in cultures maintained with sediment (Table
1). Of the 17 PCB congeners tested, only 2,3,4-CB, 2,3,4,6-CB, and 2,3,4,5,6-CB were meta
dechlorinated to 2,3-CB, 2,4,6-CB, and 2,3,5,6-CB (or 2,3,4,6-CB),
respectively. Congener 3,4,5-CB was para dechlorinated to
3,5-CB. None of the other congeners that contained at least one
meta or para chlorine were dechlorinated. The
results indicate that the 2,3,4,5-CB enrichment culture will remove
only chlorines that are flanked on each side (doubly flanked) by other
chlorines regardless of meta or para positioning.
No difference in specificity was observed between the sediment and
sediment-free enrichment cultures (Table 1). However, the extent of
dechlorination was greater with sediment, especially with the congeners
2,4- and 2,4,6-CB. These results suggest that the sediment-free
cultures selectively dechlorinate doubly flanked para
chlorines.
Effects of inhibitors and antibiotics.
The dechlorination by
the sediment-free 2,3,4,5-CB enrichment culture was also tested in the
presence of bromoethanesulfonate (BES), molybdate, ampicillin,
chloramphenicol, neomycin, streptomycin, and vancomycin. Two to ten
millimolar sodium molybdate and 100 µg of ampicillin,
chloramphenicol, neomycin, or streptomycin per ml inhibited the
dechlorination activity and the growth of this consortium (data not
shown). Sodium molybdate inhibits the growth of sulfate-reducing
microorganisms but can also cause nonspecific inhibitions
(12). The mechanism of the molybdate inhibition observed
here was not examined further. Dechlorination of 2,3,4,5-CB was
observed in the presence of 2 mM BES or 100 µg of vancomycin per ml,
although the rate was lower (Fig. 5). These results are consistent with
the absence of methanogenesis and further confirm that methanogens are
not required for the dechlorination activities observed.
| |
DISCUSSION |
A 2,3,4,5-CB para-dechlorinating consortium was
enriched from Charleston Harbor sediment. It could be transferred and
sustained in sediment-free minimal medium containing 2,3,4,5-CB and
fumarate. This is only the second PCB-dechlorinating consortium shown
to maintain its dechlorinating activity in a defined medium without the
addition of sediment. In contrast to the first sediment-free culture
(8), this enrichment culture cannot remove ortho
chlorines but can dechlorinate meta and para
chlorines. However, this PCB-dechlorinating consortium attacks only
doubly flanked chlorines of PCBs, and once established under
sediment-free conditions, it selectively dechlorinates doubly flanked
para chlorines.
Microbial PCB dechlorination has most often been observed as the
removal of meta and para chlorines of PCBs
(2), although there have been a few reported cases of
ortho dechlorination under anaerobic conditions (5, 8,
15, 18, 19). It has been proposed that discrete dechlorinating
microorganisms with distinct dehalogenating enzymes and congener
regiospecificities are responsible for the various dechlorination
processes (2-4, 6, 7, 13). However, this is only the second
report of an anaerobic culture expressing a highly specific PCB
dechlorination in a defined medium.
The 2,3,4,5-CB-dechlorinating consortium enriched from Charleston
Harbor sediment para dechlorinated 2,3,4,5-CB to 2,3,5-CB, while meta dechlorination of 2,3,4,5-CB to 2,4,5-CB was lost
after two transfers. Recently we have enriched for a strict
meta-dechlorinating culture from the same Charleston Harbor
sediment using 2,3,5,6-CB (our unpublished data). In contrast to the
doubly flanked dechlorinating culture described here, the
meta-dechlorinating culture does not para
dechlorinate 2,3,4,5-CB and does not require flanking of the target
chlorine. Cutter et al. were able to select for ortho dechlorination of 2,3,5,6-CB following transfer of a meta-
and ortho-dechlorinating enrichment culture developed with
Baltimore Harbor sediment (8). These results show that
selection for specific dechlorinating activities is dependent upon the
choice of congeners and inoculum source. Such an observation is
consistent with the hypothesis that distinct consortia or individual
species of microorganisms are responsible for specific dechlorination activities.
The combination of highly dilute, frequent transfers (0.1 to 1.0%,
vol/vol) and transfer made immediately after 50% of the 2,3,4,5-CB had
been para dechlorinated likely led to the selection of a
nonmethanogenic, highly specific dechlorinating culture. Such
conditions would probably not allow for the proliferation of
slow-growing methanogens, and early transfer may have minimized the
growth of species that would dechlorinate secondary congener products.
The lack of inhibition due to BES, an inhibitor of methanogenesis, is
not surprising in light of the culture being nonmethanogenic. However,
PCB dechlorination is usually observed under methanogenic conditions
(1, 10, 11, 14, 21, 22), and BES has been shown to inhibit
dechlorination of certain PCB congeners (14, 16) or
dechlorination processes (11). Such inhibition has been
hypothesized to be due to the dechlorinating microorganisms using the
sulfonic acid moiety of the BES, instead of a PCB, as an electron
acceptor (23). The results presented here suggest that BES
does not competitively inhibit the dechlorination of doubly flanked
chlorines of PCBs by the microorganisms in this culture. Other examples
of PCB dechlorination in the absence of methanogenesis include the
meta dechlorination of Aroclor 1242 by pasteurized
microorganisms (21) and the meta and
para dechlorination of a mixture of congeners following
growth of a mixed bacterial population on agar medium (10).
The combined effect of discrete PCB-dechlorinating pathways contributes
to the overall dechlorination of Aroclors, and such combinations are
referred to as dechlorination processes (2). There are at
least five processes (Q, H', H, P, and LP) that involve para
dechlorination of PCBs (2, 4). Process Q removes virtually all para chlorines, regardless of the surrounding chlorine
configuration, and meta chlorines of 2,3- and possibly
2,3,6-chlorophenyl groups. Process H' is characterized by the removal
of para chlorines in 3,4- and 2,4,5-chlorophenyl and
meta chlorines in 2,3-, 2,3,4-, and 2,3,6-chlorophenyl.
Process H is very much like H' except that there is no dechlorination
of 2,3-chlorophenyl groups. Process LP dechlorinates PCBs by removal of
unflanked para chlorines of PCBs. Process P dechlorinates
flanked and doubly flanked para-substituted PCBs containing
3,4-, 2,3,4-, 2,4,5-, and 2,3,4,5-chlorophenyl groups. The specific
dechlorination activity of the 2,3,4,5-CB para-dechlorinating culture described here does not match
any of these para-dechlorinating processes, but the
microbial catalysts within this culture may contribute to process P. The dechlorination of Aroclor 1260 with Charleston Harbor sediment most
closely resembles process N plus some process P. Process N is
characterized by an almost exclusive loss of flanked and doubly flanked
meta chlorines (2, 13). It is conceivable that
the microorganisms belonging to the culture described here contributed
to the Aroclor dechlorination reported in Fig. 1. However, it is
impossible to document a highly specific activity such as the
requirement of doubly flanked chlorines within an Aroclor mixture of
congeners. Only after sequential transfers in the presence of the
single congener, i.e., 2,3,4,5-CB, which stimulated the para
dechlorination of Aroclor 1260, could this highly specific activity be characterized.
In summary, a PCB-dechlorinating consortium is sustainable in a
defined, sediment-free medium and exhibits a very selective dechlorination activity. This is the first microbial consortium shown
to para or meta dechlorinate a PCB congener in a
defined and sediment-free medium. Furthermore, the culture exhibits a highly specific form of PCB dechlorination, namely, that of only doubly
flanked chlorines, while favoring the para position. This is
only the second report of a PCB-dechlorinating culture in a defined
minimal medium. The culture clearly possesses a specific dechlorinating
activity distinct from that of the other sediment-free PCB-dechlorinating culture, i.e., doubly flanked para or
meta dechlorination versus ortho dechlorination.
| |
ACKNOWLEDGMENTS |
The work was supported by the Office of Naval Research, U.S.
Department of Defense (grant N00014-99-1-0978 to H.D.M. and grant N00014-99-1-0101 to K.R.S.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Medical
University of South Carolina, Department of Microbiology and
Immunology, 173 Ashley Ave., 225 BSB, P.O. Box 250504, Charleston, SC
29425-2230. Phone: (843) 792-7140. Fax: (843) 792-2464. E-mail:
MAYH{at}MUSC.EDU.
| |
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Applied and Environmental Microbiology, January 2000, p. 49-53, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
