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Applied and Environmental Microbiology, June 1999, p. 2547-2552, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Degradation of Chlorobenzenes at Nanomolar Concentrations by
Burkholderia sp. Strain PS14 in Liquid Cultures and in
Soil
Peter
Rapp* and
Kenneth N.
Timmis
Division of Microbiology, GBF-National
Research Centre for Biotechnology, Braunschweig, Germany
Received 29 December 1998/Accepted 9 April 1999
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ABSTRACT |
The utilization of 1,2,4,5-tetrachloro-, 1,2,4-trichloro-, the
three isomeric dichlorobenzenes and fructose as the sole carbon and
energy sources at nanomolar concentrations was studied in batch
experiments with Burkholderia sp. strain PS14. In liquid culture, all chlorobenzenes were metabolized within 1 h from their initial concentration of 500 nM to below their detection limits of 0.5 nM for 1,2,4,5-tetrachloro- and 1,2,4-trichlorobenzene and 7.5 nM for
the three dichlorobenzene isomers, with 63% mineralization of the
tetra- and trichloroisomers. Fructose at the same initial concentration
was, in contrast, metabolized over a 4-h incubation period down to a
residual concentration of approximately 125 nM with 38% mineralization
during this time. In soil microcosms, Burkholderia sp.
strain PS14 metabolized tetrachlorobenzene present at 64.8 ppb and
trichlorobenzene present at 54.4 ppb over a 72-h incubation period to
below the detection limits of 0.108 and 0.09 ppb, respectively, with
approximately 80% mineralization. A high sorptive capacity of
Burkholderia sp. strain PS14 for 1,2,4,5-tetrachlorobenzene was found at very low cell density. The results demonstrate that Burkholderia sp. strain PS14 exhibits a very high affinity
for chlorobenzenes at nanomolar concentrations.
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INTRODUCTION |
Chlorinated benzenes are widely used
as solvents in chemical reactions and to dissolve materials, such as
oils, waxes, resins, greases, and rubber, and are also employed in the
production of herbicides and pesticides. They are highly toxic
compounds that cause a wide variety of effects ranging from
immunological disorders to adverse effects on the liver, kidney,
thyroid, and lung, sometimes accompanied by porphyria (13,
14). Large amounts have been produced (e.g., in 1986 17,000 to
20,000 tons of 1,2-dichlorobenzene [1,2-DCB] and 25,000 tons of
1,4-dichlorobenzene [1,4-DCB] and in 1985 5,000 tons of
1,2,4-trichlorobenzene [1,2,4-TCB] were produced in the Federal
Republic of Germany [27]), and much of this has
entered the environment. Such compounds also escape in drainage
fluids from hazardous waste disposal sites. Although the amounts
of chlorinated benzenes present in contaminated soils and sediments can
be high, their concentrations in the aqueous phase of such materials
are rather low, often in the micro- to nanomolar range, due to their
poor water solubility and sorption to colloid particles in the surface
waters or to the matrix or organic phase of the soil (45).
Only oligotrophic or facultatively oligotrophic microorganisms can grow
on substrates at such low concentrations (25, 35), which
makes these microorganisms particularly suitable for the degradation of
poorly bioavailable pollutants. A number of studies have shown that
certain pollutant and nonpollutant substrates are only degraded to some
residual concentrations, after which no further metabolism is observed (2, 11, 19, 28, 31, 32, 42, 43). Various explanations of
such threshold concentrations have been suggested, including the
possibility that they represent the lowest substrate concentration needed to maintain viability of the microorganism and the notion that
they are below the level needed to maintain synthesis of the catabolic
enzymes. Such thresholds are not absolute but vary with the compound to
be degraded and the degrading bacterium and its environment. The
existence of threshold concentrations may account for the persistence
of degradable organic compounds in the environment and may have
implications for the removal of contaminants from soil, sediments, and groundwater.
Burkholderia sp. strain PS14 (formerly known as
Pseudomonas sp. strain PS14 (29) but now
reclassified by 16S rRNA analysis (21a) is, with
Burkholderia sp. strain PS12 (5), the only microorganism presently known to utilize 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB) aerobically as the sole source of carbon and
energy (29). Burkholderia sp. strain PS14 also
degrades 1,2,4-TCB and the three isomeric dichlorobenzenes
(1,2-DCB, 1,3-DCB, and 1,4-DCB) as do some other organisms
(10, 12, 16, 17, 22, 23, 29, 34, 36-38, 43, 44, 49).
Chlorinated benzenes are converted by PS12 and PS14 by an aromatic ring
dioxygenase and a dihydrodiol dehydrogenase to chlorocatechols as
central intermediates (5, 29), which are degraded via
the modified ortho-cleavage pathway to 3-oxoadipate
(29).
The novelty of the catabolic ability of strain PS14 and its potential
importance for degradation of highly chlorinated benzenes in the
environment led us to investigate its ability to degrade chlorinated benzenes at nanomolar concentrations in liquid and in
soil cultures to determine possible threshold concentrations in
these systems. Fructose was chosen as a control substrate to compare the rate and extent of degradation of hydrophobic
chlorobenzenes with those of a hydrophilic compound, since from all the
mono- and disaccharides tested, including the pentoses and hexoses of the polysaccharides of the organic soil fraction (1), it was the only one PS14 was able to grow on. In this report we present results which show rapid degradation of chlorobenzene isomers by PS14
and indicate that any threshold concentrations for the metabolism of
chlorobenzenes at nanomolar concentrations are below current analytical
detection limits.
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MATERIALS AND METHODS |
Chemicals.
1,2-, 1,3-, and 1,4-DCB were obtained from Merck
(Darmstadt, Germany). 1,2,4-TCB was from Fluka (Neu-Ulm, Germany).
1,2,4,5-TeCB was supplied by Aldrich (Steinheim, Germany). Yeast
extract and tryptone were purchased from Difco (Detroit, Mich.).
Substrates uniformly labelled with 14C had the following
specific activities: 1,2,4-TCB, 21 mCi/mmol; D-fructose,
285 mCi/mmol; and 1,2,4,5-TeCB, 13.2 mCi/mmol); 1,2,4-TCB (98.9%
purity) and D-fructose (98.1% purity) were from Amersham (Little Chalfont, Buckinghamshire, England), and 1,2,4,5-TeCB (99%
purity) was from Sigma (St. Louis, Mo.). All other reagents were
analytical grade from commercial sources. The solubilities of these
chlorinated benzenes at 20°C in water are as follows: 1,2-DCB,
1,020.0 µM (4); 1,3-DCB, 7,482.5 µM (4);
1,4-DCB, 476.2 µM (4); 1,2,4-TCB, 165.3 µM
(27); and 1,2,4,5-TeCB, 2.5 µM (48).
Growth conditions.
Mineral salts medium had the
following composition (in grams per liter of distilled water):
Na2HPO4 · 2H2O, 7.0;
KH2PO4, 2.0; (NH4)2SO4, 0.5;
MgCl2 · 6H2O, 0.1;
Ca(NO3)2 · 4H2O, 0.05; and 1 ml of a trace element solution (24) but without EDTA
(29). Inocula were grown in 500-ml shake flasks containing
100 ml of mineral salts medium on a rotary shaker (120 rpm) at 30°C.
Finely mortar-ground 1,2,4,5-TeCB was added after sterilization to an initial concentration equivalent to 1 mM, whereas 1,2,4-TCB and the
three isomeric dichlorobenzenes were added via the vapor phase to an
initial concentration of 0.2 mM. Fructose, dissolved in mineral salts
medium, was filter sterilized and added to a final concentration of
27.8 mM. The inocula were grown, until the cells reached approximately
the middle of the logarithmic growth phase. Starved inocula prepared
from fructose-grown cultures were obtained by incubating washed cells
in mineral salts medium without a carbon source for 24 h. The
inocula grown on the different carbon sources were centrifuged at
12,000 × g for 20 min at 4°C. Prior to
centrifugation, inocula with 1,2,4,5-TeCB as the carbon source were
aseptically freed from undissolved TeCB by filtration. The pellets were
aseptically washed two times with mineral salts medium, resuspended in
a small volume of mineral salts medium, and incubated for 3 h at
ambient temperature to allow the intracellular substrate and any
substrate still adhering to the cells to be degraded. After
centrifugation, the pellet was resuspended to an optical density at 578 nm of 0.015 (corresponding to approximately 106 CFU/ml or
6.7 mg [dry weight] of bacteria per liter) in 100 ml of fresh mineral
salts medium containing the corresponding chlorobenzenes or fructose at
a concentration of 500 nM. Prior to use of the culture media, several
aliquots were taken and the substrate content was quantitated. For
determination of residual fructose concentration, a mineral salts
medium with reduced salt content and having the following composition
(in grams per liter of distilled water): Na2HPO4 · 2H2O, 0.1;
(NH4)2SO4, 0.03;
MgCl2 · 6H2O, 0.01;
Ca(NO3)2 · 4H2O, 0.01; and 1 ml of a trace element solution as described above was used. Incubations
were carried out in tightly closed 500-ml flasks at 30°C with
shaking. Controls of uninoculated media were made. Bacteria were
enumerated on solid medium composed of 10 g of tryptone, 5 g
of yeast extract, 10 g of NaCl, and 15 g of agar per liter.
For analyzing the degradation of chlorobenzenes in soil, a
low-contaminated soil (BBA standard soil, Borstel near Neustadt am
Rübenberge, Lower Saxonia, Germany) was used; this soil is recommended by the Biological Federal Institute for Agriculture and
Forestry (BBA) in Braunschweig, Germany, as a standard soil for testing
of herbicides for approval (30). It was composed of 74.2%
(wt/wt) sand, 19.5% (wt/wt) silt, and 6.3% (wt/wt) clay. Its organic
C content was 1.31% (wt/wt), its pH was 6.0, and its maximal water
capacity was 36% (wt/wt). The soil was passed through a
2-mm-pore-diameter sieve. Nonsterile soil was dried in a desiccator with silica gel for 3 days in order to achieve good distribution of the
chlorinated benzenes, whereas sterilized soil had been autoclaved three
times (45 min at 121°C) and dried. 1,2,4,5-TeCB or 1,2,4-TCB
dissolved in 17 ml of mineral salts medium was thoroughly mixed into
83 g of soil with a spatula to give a 1,2,4,5-TeCB concentration
of 64.8 ppb or a 1,2,4-TCB concentration of 54.4 ppb. Both
concentrations correspond to 300 nmol/kg (wet weight) of soil. The soil
was inoculated with amounts of Burkholderia sp. strain PS14
which gave an optical density at 578 nm of 0.015 in a liquid culture of
the same weight (corresponding to approximately 106 CFU/ml
or 6.7 mg [dry weight] of bacteria per liter). The degradation studies were carried out in tightly closed 500-ml shake flasks at
30°C at 120 rpm. For enumeration of the bacteria, 1 g of sterile soil mixed with 1,2,4,5-TeCB or 1,2,4-TCB and inoculated with Burkholderia sp. strain PS14 was suspended in 99 ml of
sodium pyrophosphate solution (2.8 g of
Na4P2O7 in 1 liter of water) and
stirred at room temperature for 5 min. The viable bacteria were
enumerated by plating 1:10 serial dilutions in triplicate.
Analytical methods.
To determine the concentration of
chlorobenzenes in liquid cultures, 100-ml shake flask cultures were
extracted twice with 50 ml of redistilled n-hexane. Soil was
extracted with 130 ml of redistilled n-hexane-acetone
(10:3, vol/vol) and twice with 100 ml of redistilled
n-hexane. The extracts were pooled, dried over anhydrous
sodium sulfate, and concentrated by evaporation to 1 ml at 1.4 × 104 Pa at 30°C. The chlorobenzenes were analyzed by gas
chromatography (model 5890; Hewlett-Packard, Wilmington, Del.) using a
63Ni high-temperature electron capture detector and a 30-m
fused silica capillary column (PTE-5; Supelco). Helium was used as a carrier gas, and nitrogen was used as a detector-quench gas. The operating temperatures of the injector and detector were 250 and 300°C, respectively. The oven temperature program was as follows: 60°C for 2 min, increase to 170°C at a rate of 4°C/min, increase to 300°C at a rate of 60°C/min, with an isothermal period of 10 min
at the end. Retention times and peak areas were determined by using the
HP 3365 ChemStation software. Peaks were identified and quantified by
comparing injections with authentic external standards prepared by
dissolving definite amounts of chlorobenzenes in mineral salts medium.
These aqueous solutions were extracted, and the extracts were
concentrated in the same way as the samples to be analyzed.
In order to ensure that the residual fructose remained completely
dissolved in small amounts of water after lyophilization
of the culture
supernatant, a mineral salts medium with a reduced
salt content was
used. A 1,000-ml culture grown on this medium
was centrifuged at
12,000 ×
g for 20 min at 4°C, and the pellet
obtained was washed with fresh medium. In order to analyze fructose
enzymatically, its binding to proteins during freeze-drying of
the
culture fluid must be prevented. Therefore, 10 µM mannose
was added
to the combined culture supernatant and washing fluid.
The culture
fluid was then stirred for 10 min at ambient temperature
and
concentrated by lyophilization to 5 ml. The concentration
of fructose
was then determined enzymatically (
6).
For tests of mineralization in liquid cultures, shake flasks containing
100 ml of mineral salts medium and the appropriate
chlorobenzene or
fructose at a concentration of 500 nM were prepared
as described above.
Approximately 10 µl of a stock of
14C-labelled
chlorinated benzenes dissolved in acetone (approximately
3 × 10
5 cpm) had previously been added, and the
solvent was allowed to
evaporate. The same amount of
14C
from a stock of
14C-labelled fructose dissolved in water
was added. The media were
inoculated with 10 to 50 µl of a cell
suspension to an optical
density at 578 nm of 0.015 (corresponding to
approximately 10
6 CFU/ml or 6.7 mg [dry weight] of
bacteria per liter). Duplicate
samples of 1 ml were acidified with 7.4 N sulfuric acid to a pH
of
2. They were stripped with air for 5 min,
mixed with 4 ml
of Ultima Gold scintillation cocktail (Packard,
Frankfurt am Main,
Germany) and the radioactivity was measured with a
liquid scintillation
counter (LS 1801; Beckman, Fullerton, Calif.). The
radioactivity
measured represents the
14C activity of
nonvolatile compounds. Preliminary experiments with
a solution of
NaH
14CO
3 of 2,800 cpm/ml which was acidified
with 7.4 N sulfuric acid
to a pH of
2 and stripped with air for
various periods demonstrated
that 99% of the labelled CO
2
was released from the solution within
5 min. Duplicate 1-ml samples
were mixed with 10 N NaOH (final
pH
12). Stripping with air and
measuring radioactivity were performed
as described above. This
measurement represents the sum of
14C activity of
CO
2 and nonvolatile compounds. The formation of
CO
2 in liquid cultures was calculated by the difference
between
the radioactivity of the alkaline and acidic samples. The
percentage
of
14C incorporation into biomass was assumed to
be the difference
between the percentage of
14C in the
untreated culture broth and the percentage of
14C in the
untreated culture
supernatant.
For tests of mineralization in soil, approximately 10 µl of a stock
of
14C-labelled 1,2,4-TCB or 1,2,4,5-TeCB dissolved in
acetone (approximately
3 × 10
5 cpm) were added to 17 ml of mineral salts medium containing the
corresponding unlabelled
substrates. This solution was thoroughly
mixed into 83 g of soil
with a spatula to give a 1,2,4-TCB concentration
of 54.4 ppb or a
1,2,4,5-TeCB concentration of 64.8 ppb. Incubation
was performed as
described above. Distilled water (50 ml) was
added to duplicate flasks,
the slurry was acidified with 7.4 N
sulfuric acid to a pH of
2 and
stirred for 10 min on a magnetic
stirrer. The treated soil slurry was
extracted once with 130 ml
of redistilled
n-hexane-acetone
(10:3, vol/vol) and two times
with 100 ml of redistilled
n-hexane. Radioactivity in 1 ml of
the extract was measured
as described above. This measurement
represents the
14C
activity of nonvolatile compounds. Preliminary experiments in
which a
soil slurry with NaH
14CO
3 (3,000 cpm/ml) was
treated similarly demonstrated that 98%
of the labelled
CO
2 was released from the soil suspension within
5 min. To
measure the
14C activity of nonvolatile compounds and
CO
2, 50 ml of distilled
water was added to a second pair of
duplicate flasks and mixed
with 10 N NaOH so that the pH of the soil
slurry was
12. The
subsequent treatment and the measurement of
radioactivity were
done as described above. The formation of
CO
2 in soil was calculated
by the difference between the
radioactivity of the extract from
the alkaline soil slurry and the
extract from the acidic soil
slurry. The losses of
14C due
to volatilization of the substrates during stirring of the
soil slurry
were very small, since the substrates were strongly
adsorbed to cells
and soil (see Results and Discussion). Sterile
controls were included
in all experiments to check for abiotic
disappearance of the
substrates.
Biosorption of 1,2,4,5-TeCB.
Washed cells of
Burkholderia sp. strain PS14 grown on fructose or
1,2,4,5-TeCB were added to 50 ml of mineral salts medium containing
0.4% (wt/vol) sodium azide to a final optical density at 578 nm of
0.03, and the suspension was shaken for 30 min at ambient temperature
and 120 rpm. Portions (50 ml) of mineral salts medium containing 0.15 to 1.4 µM 1,2,4,5-TeCB were then added so that the cell suspension
obtained had a concentration of 6.7 mg (dry weight) of bacteria per
liter. After 1, 3, 5, 10, 20, 30, and 60 min, 100-ml samples of this
cell suspension were rapidly centrifuged at 12,000 × g
at 4°C and the supernatant was analyzed for 1,2,4,5-TeCB as described
above. Controls were uninoculated mineral salts medium also containing
0.2% (wt/vol) sodium azide and 1,2,4,5-TeCB of the corresponding
concentration. The recovery efficiency for 1,2,4,5-TeCB from autoclaved
cell suspensions and cell suspensions treated with sodium azide ranged
from 91 to 98%.
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RESULTS |
Degradation of chlorobenzenes and fructose in liquid cultures.
In liquid cultures of Burkholderia sp. strain PS14 with
1,2,4,5-TeCB or 1,2,4-TCB as the only carbon and energy source, the chlorobenzene concentration decreased during 1 h of incubation from 500 nM to below the detection limit of 0.5 nM. In cultures containing radioactive substrate, about 63% of the added radiolabel was converted to CO2 (Fig.
1A). Approximately 26% of
14C from 1,2,4,5-TeCB was incorporated into biomass over a
5-h period of incubation. When 1,2,4,5-TeCB and 1,2,4-TCB were used
together at initial concentrations of 250 nM, both substrates were
metabolized simultaneously and as rapidly as if they were the sole
substrate. Similarly, 1,2-DCB, 1,3-DCB, and 1,4-DCB were metabolized
within 1 h from initial concentrations of 500 nM to below their
detection limits of 7.5 nM (data not shown).
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FIG. 1.
Metabolism and mineralization of 1,2,4,5-TeCB,
1,2,4-TCB, and fructose in liquid cultures of Burkholderia
sp. strain PS14 in mineral salts medium. Percentages of mineralization
(solid symbols) and nanomolar concentrations (open symbols) of
1,2,4,5-TeCB, 1,2,4-TCB, and fructose are shown. (A)  ,
[14C]1,2,4,5-TeCB;  , [14C]1,2,4-TCB;
 , 1,2,4,5-TeCB;  , 1,2,4-TCB;  , 1,2,4-TCB in the sterile
control. (B) , [14C]fructose; , fructose. The
values are the means of results of two to four independent experiments,
and error bars show standard deviations.
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In contrast, fructose was metabolized much more slowly and only to a
threshold of approximately 125 nM during a 4-h incubation
(Fig.
1B). No
difference in the residual fructose concentration
between cultures
inoculated with starved or nonstarved cells was
found. Approximately
38% of fructose had been mineralized and
approximately 31% had been
incorporated into biomass after 4 h
of incubation. Cell densities
at 578 nm of about 0.015 and the
number of viable cells of
approximately one million CFU of PS14
per ml on 500 nM chlorinated
benzenes or fructose did not increase
significantly.
Metabolism and mineralization of 1,2,4,5-TeCB and 1,2,4-TCB in
soil.
Metabolism and mineralization of 64.8 ppb of 1,2,4,5-TeCB
(corresponding to 300 nmol/kg [wet weight] of soil) were measured in
both sterilized and nonsterilized soil having a moisture content of
20% (wt/wt) inoculated with Burkholderia sp. strain PS14.
In both soils, the concentration of 1,2,4,5-TeCB fell to below the detection limit of 0.108 ppb (corresponding to 0.5 nmol/kg [wet weight] of soil) during 72 to 96 h of incubation. During this period approximately 80% of the substrate was mineralized in both soils and the number of viable cells of PS14 increased from 7.6 × 105 to 647 × 105 per g (wet weight) of
nonsterilized soil (Fig. 2). Similar
results were obtained with 1,2,4-TCB as the substrate (Fig.
3). No degradation was observed in
uninoculated soils. This and the results with 1,2,4,5-TeCB indicate
that indigenous soil microorganisms or sterilization (autoclaving) of
the soil did not influence the degradation and mineralization of
1,2,4,5-TeCB by PS14.
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FIG. 2.
Metabolism and mineralization of 1,2,4,5-TeCB in
unsterilized and sterilized BBA standard soil inoculated with
Burkholderia sp. strain PS14. Percentages of mineralization
of [14C]1,2,4,5-TeCB (solid symbols) and concentrations
(in parts per billion) of 1,2,4,5-TeCB (open symbols) in nonsterile
(circles) and sterile (squares) soil are shown.  , concentrations (in
parts per billion) of 1,2,4,5-TeCB in the uninoculated control; ,
CFU of Burkholderia sp. strain PS14 per gram (wet weight) of
sterile soil. The values are the means of results of two to four
independent experiments, and error bars show standard deviations.
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FIG. 3.
Metabolism and mineralization of 1,2,4-TCB in sterilized
BBA standard soil inoculated with Burkholderia sp. strain
PS14. Percentages of mineralization of [14C]1,2,4-TCB
() and concentrations of 1,2,4-TCB (in parts per billion) () in
soil are shown. , concentration (in parts per billion) of 1,2,4-TCB
in the uninoculated control; , CFU per gram (wet weight) of soil.
The values are the means of results of four independent experiments,
and the error bars show standard deviations.
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Sorption of 1,2,4,5-TeCB onto PS14 cells.
Sorption of
1,2,4,5-TeCB onto cells of Burkholderia sp. strain PS14 was
studied with autoclaved and 0.2% (wt/vol) sodium azide-containing cell
suspensions. Their concentrations were 6.7 mg (dry weight) of bacteria
per liter corresponding to an optical density at 578 nm of 0.015. Sorption reached equilibrium within 3 to 5 min. Eighty-three percent of
0.5 µM 1,2,4,5-TeCB was adsorbed on cells treated with sodium azide.
Autoclaved cells showed the same sorptive capacity for 1,2,4,5-TeCB
(82.5% ± 2.6%) as those treated with sodium azide. There was no
difference in sorption of 1,2,4,5-TeCB between cells of PS14 grown on
fructose (82.8% ± 2.5%) or 1,2,4,5-TeCB (83% ± 1.1%). An isotherm
was determined for the biosorption of 1,2,4,5-TeCB onto cells of PS14
treated with 0.2% (wt/vol) sodium azide (Fig. 4). Linear regression of these data
yielded a partition coefficient (Kp) of
7.18 × 105 ± 0.93 × 105
cm3/g (dry weight) of cells.
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FIG. 4.
Linear isotherm for 1,2,4,5-TeCB sorption to
Burkholderia sp. strain PS14. The values represent the means
of duplicate samples.
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DISCUSSION |
The ability to aerobically degrade 1,2,4,5-TeCB, in addition to
the less-chlorinated congeners (29), makes
Burkholderia sp. strain PS14 a potentially important
resource for bioremediation of chlorobenzene-contaminated sites. A
critical requirement of remediation processes is reduction of
pollutants to below prescribed levels. The applicability of
bioremediation is sometimes limited by substrate threshold levels which
determine the extent to which a pollutant can be degraded. For this
reason, we have analyzed the degradation of chlorobenzenes present at
very low concentrations by strain PS14. The degradation of
1,2,4,5-TeCB, 1,2,4-TCB, the three dichlorobenzene isomers, and
fructose as the sole carbon and energy sources in liquid batch cultures
of PS14 was studied at concentrations in the nanomolar range. The
concentration of the chlorinated benzenes decreased from 500 nM to
below the detection limits within 1 h. This suggests that in batch
experiments with PS14 under the described conditions no measurable
residual concentrations (threshold values) exist for these compounds.
Such a phenomenon was also observed by Tros et al. (40) for
the degradation of 3-chlorobenzoate in batch cultures of
Pseudomonas sp. strain B13 and by van der Meer et al.
(43) during a batch incubation of an effluent from a soil
column continuously fed with 1,2-DCB. On the other hand, distinct
threshold concentrations for substrate consumption have been observed
for many other bacteria (7, 11, 18, 19, 21, 28, 32, 47).
Such residual concentrations were correlated with the maintenance needs
of the microorganisms (28, 33). Bosma et al. (8)
introduced the mass transfer of a chemical to the degrading
microorganism as a second parameter determining the occurrence and
height of a threshold value and established a relationship in which the
threshold concentration of a compound for growth is inversely
proportional to the exchange constant k, a measure of mass
transfer, and directly proportional to the base-level maintenance flux
qm. According to this model, our failure to
detect threshold values for chlorinated benzenes above the
corresponding detection limits in batch cultures of PS14 implies a very
low maintenance requirement of the nongrowing cells and a nonlimiting
mass transfer.
In contrast to the metabolism of chlorinated benzenes, fructose at an
initial concentration of 500 nM was metabolized more slowly and
incompletely, so that a residual concentration of approximately 125 nmol/liter remained after 4 h of incubation, regardless of whether
the cells had been starved or not. Only 38% of the fructose was
mineralized, and approximately 31% of [14C]fructose was
incorporated into biomass during the 4-h incubation (63% of the
1,2,4,5-TeCB was mineralized within 1 h of incubation, and
approximately 26% of 14C was incorporated into biomass).
The PS14 strain is thus clearly specialized in its metabolism for
chlorobenzenes and does not catabolize the hydrophilic control
substrate fructose efficiently.
Although 1,2,4,5-TeCB and 1,2,4-TCB were degraded by strain PS14 to
below the detection limits in both liquid cultures and soil, they were,
as expected, metabolized and mineralized faster in liquid cultures,
presumably due to their adsorption to soil particles. In the absence of
PS14, 83.5% of 255 nM 1,2,4,5-TeCB adsorbed to 83 g of soil/liter
used in the present study within a 30-min incubation, which corresponds
to a Kp of 61 cm3/g. Only 14.3% of
this sorbed 1,2,4,5-TeCB could be desorbed by exchanging the aqueous
phase (20). Clearly, PS14 must enhance desorption of
1,2,4,5-TeCB from soil and/or effectively compete by adsorbing this
compound more efficiently than soil. Our experiments suggest that
1,2,4,5-TeCB adsorbs to PS14 with a Kp of
7.18 × 105 cm3/g, i.e., approximately 4 orders of magnitude more strongly than to the soil used in the present
study. Using the same low concentration of cells as in the degradation
experiments but autoclaved or treated with sodium azide, approximately
83% of 500 nM 1,2,4,5-TeCB adsorbed within 3 to 5 min. There was no
difference in sorption between fructose- and 1,2,4,5-TeCB-grown cells.
Sorption of 1,2,4,5-TeCB to PS14 seemed to be passive, since dead cells
sorbed as well as live cells (data not shown; see also references
41 and 46). The
Kp of 1,2,4,5-TeCB in suspensions of PS14 cells
was 7.18 × 105 ± 0.93 × 105
cm3/g, namely, about the same order of magnitude as the
octanol-water partition coefficient (Kow) of
1,2,4,5-TeCB of 1.12 × 105 (48). The
Kp of 1,2,4,5-TeCB in suspensions of PS14 cells
was thus about 40 times greater than that predicted by the empirical relation log Kp = 0.907 log
Kow 
0.361 established by Baughman and
Paris (3) between bacterial Kp and
the Kow of organic compounds. This difference
may be partly due to the extremely low biomass of 6.7 mg (dry weight)
of cells per liter used in our sorption studies, since it has been
shown by Brandt et al. (9) that the sorption capacity of a
bacterium for a highly chlorinated organic compound significantly
increases with decreasing biomass in the concentration range below 0.5 g/liter. In contrast to the degradation of 1,2,4-TCB and 1,2,4,5-TeCB
by PS14 to below detection limits in a soil contaminated for 1 or 2 weeks with these chemicals, residual concentrations of these compounds
were found in batch cultures of a mixed population and a soil
contaminated for some decades with chlorinated benzenes
(15). This incomplete degradation may be attributed partly
to transformation systems with lower affinities for chlorobenzenes than
those in PS14. Nevertheless, the progressive entrapment of 1,2,4-TCB
and 1,2,4,5-TeCB in microscopic pores of the soil is almost certainly
the most important barrier to complete degradation (39).
However, in the case of PS14, the large differences in partition
coefficients of soil and PS14 cells for chlorobenzenes will result in
steep concentration gradients in soil that promote faster intraparticle
mass transfer rates (26) than those which would exist in the
absence of PS14 cells. This would obviously be an important property
for an organism that is to be used for bioremediation.
| |
ACKNOWLEDGMENTS |
We thank M. Sylla for technical assistance and Fritz Homann
(Borstel, Neustadt am Rübenberge, Germany) for supplying the BBA
standard soil. We are grateful to E. R. B. Moore for the 16S rRNA analysis and to D. H. Pieper and R.-M. Wittich for valuable discussions.
This research was financially supported by the German Federal Ministry
for Education, Science, Research and Technology (BMBF grant 0139433).
Kenneth N. Timmis expresses gratitude to the Fonds der Chemischen
Industrie for generous support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Microbiology, GBF-National Research Centre for Biotechnology,
Mascheroderweg 1, D-38124 Braunschweig, Germany. Phone:
49-(0)531/6181-468. Fax: 49-(0)531/6181-411. E-mail:
pra{at}gbf.de.
| |
REFERENCES |
| 1.
|
Alexander, M.
1977.
The soil environment, p. 3-15.
In
M. Alexander (ed.), Introduction to soil microbiology. John Wiley & Sons, New York, N.Y.
|
| 2.
|
Alexander, M.
1985.
Biodegradation of organic chemicals.
Environ. Sci. Technol.
18:106-111.
|
| 3.
|
Baughman, G. L., and D. F. Paris.
1981.
Microbial bioconcentration of organic pollutants from aquatic systems a critical review.
Crit. Rev. Microbiol.
8:205-228[Medline].
|
| 4.
|
Beck, U.
1986.
Nucleus-chlorinated aromatic hydrocarbons, p. 330-355.
In
F. T. Campbell, R. Pfefferkorn, and J. F. Rounsaville (ed.), Ullmann's encyclopedia of industrial chemistry, 5th ed. VCH Verlagsgesellschaft, Weinheim, Germany.
|
| 5.
|
Beil, S.,
B. Happe,
K. N. Timmis, and D. H. Pieper.
1997.
Genetic and biochemical characterization of the broad spectrum chlorobenzene dioxygenase from Burkholderia sp. strain PS12. Dechlorination of 1,2,4,5-tetrachlorobenzene.
Eur. J. Biochem.
247:190-199[Medline].
|
| 6.
|
Boehringer Mannheim Biochemica.
1994.
Methoden der enzymatischen Bioanalytik und Lebensmittelanalytik.
Boehringer Mannheim, Mannheim, Germany.
|
| 7.
|
Boethling, R. S., and M. Alexander.
1979.
Effect of concentration of organic chemicals on their biodegradation by natural microbial communities.
Appl. Environ. Microbiol.
37:1211-1216[Abstract/Free Full Text].
|
| 8.
|
Bosma, T. N. P.,
P. J. M. Middeldorp,
G. Schraa, and A. J. B. Zehnder.
1997.
Mass transfer limitation of biotransformation: quantifying bioavailability.
Environ. Sci. Technol.
31:248-252.
|
| 9.
|
Brandt, S.,
A.-P. Zeng, and W.-D. Deckwer.
1997.
Adsorption and desorption of pentachlorophenol on cells of Mycobacterium chlorophenolicum PCP-1.
Biotechnol. Bioeng.
55:480-489.
|
| 10.
|
Brunsbach, F. R., and W. Reineke.
1994.
Degradation of chlorobenzenes in soil slurry by a specialized organism.
Appl. Microbiol. Biotechnol.
42:415-420[Medline].
|
| 11.
|
Button, D. K.
1985.
Kinetics of nutrient-limited transport and microbial growth.
Microbiol. Rev.
49:270-297[Free Full Text].
|
| 12.
|
De Bont, J. A. M.,
M. J. A. W. Vorage,
S. Hartmans, and W. J. J. van den Tweel.
1986.
Microbial degradation of 1,3-dichlorobenzene.
Appl. Environ. Microbiol.
52:677-680[Abstract/Free Full Text].
|
| 13.
|
Den Besten, C.,
J. J. R. M. Vet,
H. T. Besselink,
G. S. Kiel,
B. J. M. van Berkel,
R. Beems, and P. J. van Bladeren.
1991.
The liver, kidney, and thyroid toxicity of chlorinated benzenes.
Toxicol. Appl. Pharmacol.
111:69-81[Medline].
|
| 14.
|
Den Besten, C.,
A. Brouwer,
I. M. C. M. Rietjens, and P. J. van Bladeren.
1994.
Biotransformation and toxicity of halogenated benzenes.
Hum. Exp. Toxicol.
13:866-875[Medline].
|
| 15.
|
Fieseler, C., and B. Noll.
1994.
Biologischer Abbau von Schadstoffen in mischkontaminierten Böden.
EntsorgungsPraxis
10:16-19.
|
| 16.
|
Haigler, B. E.,
S. F. Nishino, and J. C. Spain.
1988.
Degradation of 1,2-dichlorobenzene by a Pseudomonas sp.
Appl. Environ. Microbiol.
54:294-301[Abstract/Free Full Text].
|
| 17.
|
Haigler, B. E.,
C. A. Pettigrew, and J. C. Spain.
1992.
Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.
Appl. Environ. Microbiol.
58:2237-2244[Abstract/Free Full Text].
|
| 18.
|
Hopkins, B. T.,
M. J. McInerney, and V. Warikoo.
1995.
Evidence for an anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader.
Appl. Environ. Microbiol.
61:526-530[Abstract].
|
| 19.
|
Jetten, M. S. M.,
A. J. M. Stams, and A. J. B. Zehnder.
1990.
Acetate threshold values and acetate activating enzymes in methanogenic bacteria.
FEMS Microbiol. Ecol.
73:339-344.
|
| 20.
|
Keskin, M.
1994.
Ph.D. thesis.
University of Hamburg, Hamburg, Germany.
|
| 21.
|
LaPat-Polasko, L. T.,
P. L. McCarty, and A. J. B. Zehnder.
1984.
Secondary substrate utilization of methylene chloride by an isolated strain of Pseudomonas sp.
Appl. Environ. Microbiol.
47:825-830[Abstract/Free Full Text].
|
| 21a.
| Moore, E. R. B. Personal communication.
|
| 22.
|
Oldenhuis, R.,
L. Kuijk,
A. Lammers,
D. B. Janssen, and B. Witholt.
1989.
Degradation of chlorinated and non-chlorinated aromatic solvents in soil suspensions by pure bacterial cultures.
Appl. Microbiol. Biotechnol.
30:211-217.
|
| 23.
|
Oltmanns, R. H.,
H. G. Rast, and W. Reineke.
1988.
Degradation of 1,4-dichlorobenzene by enriched and constructed bacteria.
Appl. Microbiol. Biotechnol.
28:609-616.
|
| 24.
|
Pfennig, N., and K. D. Lippert.
1966.
Über das Vitamin B12-Bedürfnis phototropher Schwefelbakterien.
Arch. Mikrobiol.
55:245-256.
|
| 25.
|
Poindexter, J. S.
1981.
Oligotrophy: fast and famine existence.
Adv. Microb. Ecol.
5:63-89.
|
| 26.
|
Rijnaarts, H. H. M.,
A. Bachmann,
J. C. Jumelet, and A. J. B. Zehnder.
1990.
Effect of desorption and intraparticle mass transfer on the aerobic biomineralization of  -hexachlorocyclohexane in a contaminated calcareous soil.
Environ. Sci. Technol.
24:1349-1354.
|
| 27.
|
Rippen, G.
1991.
Handbuch Umweltchemikalien.
Ecomed Verlagsgesellschaft AG & Co KG, Landsberg am Lech, Germany.
|
| 28.
|
Rittmann, B. E., and P. L. McCarty.
1980.
Evaluation of steady-state-biofilm kinetics.
Biotechnol. Bioeng.
22:2359-2373.
|
| 29.
|
Sander, P.,
R.-M. Wittich,
P. Fortnagel,
H. Wilkes, and W. Francke.
1991.
Degradation of 1,2,4-trichloro- and 1,2,4,5-tetrachlorobenzene by Pseudomonas strains.
Appl. Environ. Microbiol.
57:1430-1440[Abstract/Free Full Text].
|
| 30.
|
Schinkel, K.
1991.
Modifizierung der Lysimeterrichtlinie (Richtlinie der Biologischen Bundesanstalt, Teil IV, 4-3).
Nachrbl. Dtsch. Pflanzenschutzd.
43:183.
|
| 31.
|
Schmidt, S. K.,
M. Alexander, and M. L. Shuler.
1985.
Predicting threshold concentrations of organic substrates for bacterial growth.
J. Theor. Biol.
114:1-8.
|
| 32.
|
Schmidt, S. K.,
K. M. Scow, and M. Alexander.
1987.
Kinetics of p-nitrophenol mineralization by a Pseudomonas sp.: effects of second substrates.
Appl. Environ. Microbiol.
53:2617-2623[Abstract/Free Full Text].
|
| 33.
|
Schmidt, S. K.,
S. Simkins, and M. Alexander.
1985.
Models for the kinetics of biodegradation of organic compounds not supporting growth.
Appl. Environ. Microbiol.
50:323-331[Abstract/Free Full Text].
|
| 34.
|
Schraa, G.,
M. L. Boone,
M. S. M. Jetten,
A. R. W. van Neerven,
P. J. Colberg, and A. J. B. Zehnder.
1986.
Degradation of 1,4-dichlorobenzene by Alcaligenes sp. strain A175.
Appl. Environ. Microbiol.
52:1374-1381[Abstract/Free Full Text].
|
| 35.
|
Semenov, A. M.
1991.
Physiological bases of oligotrophy of microorganisms and the concept of microbial community.
Microb. Ecol.
22:239-247.
|
| 36.
|
Spain, J. C., and S. F. Nishino.
1987.
Degradation of 1,4-dichlorobenzene by a Pseudomonas sp.
Appl. Environ. Microbiol.
53:1010-1019[Abstract/Free Full Text].
|
| 37.
|
Spiess, E.,
C. Sommer, and H. Görisch.
1995.
Degradation of 1,4-dichlorobenzene by Xanthobacter flavus 14p1.
Appl. Environ. Microbiol.
61:3884-3888[Abstract].
|
| 38.
|
Springer, W., and H. G. Rast.
1988.
Biologischer Abbau mehrfach halogenierter mono- und polyzyklischer Aromaten.
GWF Wasser Abwasser
129:70-75.
|
| 39.
|
Steinberg, S. M.,
J. J. Pignatello, and B. L. Sawhney.
1987.
Persistence of 1,2-dibromoethane in soils: entrapment in intraparticle micropores.
Environ. Sci. Technol.
21:1201-1208.
|
| 40.
|
Tros, M. E.,
G. Schraa, and A. J. B. Zehnder.
1994.
Biodegradation of 3-chlorobenzoate at low concentrations: transformation kinetics and residual concentrations, p. 70-72.
In
IchemE Second International Symposium on Environmental Biotechnology. Chameleon Press Ltd., London, United Kingdom.
|
| 41.
|
Tsezos, M., and J. P. Bell.
1989.
Comparison of the biosorption and desorption of hazardous organic pollutants by live and dead biomass.
Water Res.
23:561-568.
|
| 42.
|
van der Kooij, D., and W. A. M. Hijnen.
1988.
Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water.
Appl. Environ. Microbiol.
54:2842-2851[Abstract/Free Full Text].
|
| 43.
|
van der Meer, J. R.,
W. Roelofsen,
G. Schraa, and A. J. B. Zehnder.
1987.
Degradation of low concentrations of dichlorobenzenes and 1,2,4-trichlorobenzene by Pseudomonas sp. strain P51 in nonsterile soil columns.
FEMS Microbiol. Ecol.
45:333-341.
|
| 44.
|
van der Meer, J. R.,
A. R. W. van Neerven,
E. J. de Vries,
W. M. de Vos, and A. J. B. Zehnder.
1991.
Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51.
J. Bacteriol.
173:6-15[Abstract/Free Full Text].
|
| 45.
|
van der Meer, J. R.,
T. N. P. Bosma,
W. P. De Bruin,
H. Harms,
C. Holliger,
H. H. M. Rijnaarts,
M. E. Tros,
G. Schraa, and A. J. B. Zehnder.
1992.
Versatility of soil column experiments to study biodegradation of halogenated compounds under environmental conditions.
Biodegradation
3:265-284.
|
| 46.
|
Wang, X., and C. P. L. Grady, Jr.
1994.
Comparison of biosorption isotherms for di-N-butyl phthalate by live and dead bacteria.
Water Res.
28:1247-1251.
|
| 47.
|
Westermann, P.,
B. K. Ahring, and R. A. Mah.
1989.
Threshold acetate concentrations for acetate catabolism by aceticlastic methanogenic bacteria.
Appl. Environ. Microbiol.
55:514-515[Abstract/Free Full Text].
|
| 48.
|
Yalkowsky, S. H., and S. C. Valvani.
1980.
Solubility and partitioning I: solubility of nonelectrolytes in water.
J. Pharm. Sci.
69:912-922[Medline].
|
| 49.
|
Zaitsev, G. M.,
J. S. Uotila,
I. V. Tsitko,
A. G. Lobanok, and M. S. Salkinoja-Salonen.
1995.
Utilization of halogenated benzenes, phenols, and benzoates by Rhodococcus opacus GM-14.
Appl. Environ. Microbiol.
61:4191-4201[Abstract].
|
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Abstract
Introduction
