Agent Orange in Vietnam and as a herbicide in the United States and other countries for defoli- ation and control of poison ivy, poison oak, and various broadleaf ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1982, p. 72-78 0099-2240/82/070072-07$02.00/0
Vol. 44, No. 1
Biodegradation of 2,4,5-Trichlorophenoxyacetic Acid by Pure Culture of Pseudomonas cepacia
a
J. J. KILBANE, D. K. CHATTERJEE, J. S. KARNS, S. T. KELLOGG, AND A. M. CHAKRABARTY* Department of Microbiology, School of Basic Medical Sciences, University of Illinois at the Medical Center, Chicago, Illinois 60612 Received 2 December 1981/Accepted 31 March 1982
A pure culture of Pseudomonas cepacia, designated AC1100, that can utilize 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as its sole source of carbon and energy was isolated. An actively growing culture of AC1100 was able to degrade more than 97% of 2,4,5-T, present at 1 mg/ml, within 6 days as determined by chloride release, gas chromatographic, and spectrophotometric analyses. The ability of AC1100 to oxidize a variety of chlorophenols and related compounds is also reported.
2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) is
grown with 2,4,5-T as a sole carbon source (10) on minimal 2,4,5-T media. Taxonomic characterization tests have demonstrated the strain to be Pseudomonas cepacia. For cultivation and growth studies, the strain was inoculated into a basal salts medium (BSM) (12) of the composition (per liter of deionized water): 5.8 g of K2HPO4, 4.5 g of KH2PO4, 2 g of (NH4)2SO4, 0.16 g of MgCl2, 20 mg of CaC12, 2 mg of NaMoO4, 1 mg of FeSO4, and 1 mg of MnCl2. 2,4,5-T was dissolved in hot water with dropwise addition of 0.1 N NaOH and was freshly made as a stock solution (25 mg/ml) from which samples were added to the medium at 1 mg/ml. The cells were incubated at 30°C with shaking, and at different times samples were withdrawn, and 2,4,5-T loss was determined by gas chromatographic or spectrophotometric means, as well as by the release of chloride ions. Samples from the aliquots were also used for determining cell density by measuring turbidity at 540 nm in a Gilford model 2600 spectrophotometer, as well as by measurement of cell survival by dilution and plating on nutrient agar plates. Gas chromatographic and spectrophotometric determination of 2,4,5-T. The amount of 2,4,5-T remaining in culture fluids was determined by direct spectrophotometric analysis at 206 or 288 nm, where UV wavelength scans of 2,4,5-T standards indicated maximal absorbance. For gas chromatographic determination of 2,4,5-T concentration, the supernatants were adjusted to pH 2 with concentrated H2SO4 and extracted three times with a 0.5 volume of ethyl acetate. The three extracts were pooled and evaporated to dryness under N2 gas. The trimethylsilyl derivatives of the dried material were prepared by suspending them in the pyridine-based solutions of N-methyl-N-trimethylsilyltrifluoroacetamide (Pierce Chemical Co.). The trimethylsilyl derivative of 2,4,5-T was resolved over an 8°C/min temperature program from 40 to 260°C in a Varian 3700 gas chromatograph equipped with a 3% OV-17 column and was detected with a flame ionization detector. Oxygen uptake studies with resting cell suspensions. 02 uptake by resting cell suspensions of AC1100 was determined directly with a Gilson 5/6 H oxygraph equipped with a Clark electrode. Endogenous respira-
a herbicide that has been used extensively for the last several decades not only for brush and weed control on rangelands, pastures, and rights-of-way, but also as a growth regulator to delay coloration of lemons, increase the size of citrus fruits, and reduce deciduous fruit drop (5). Extensive use of 2,4,5-T as a component of Agent Orange in Vietnam and as a herbicide in the United States and other countries for defoliation and control of poison ivy, poison oak, and various broadleaf weeds has created toxicological problems (6), leading to a restricted usage of this compound in the United States and other countries (4). The persistence of 2,4,5-T has contributed significantly to this pollution problem, since this compound is known to be biodegraded very slowly and only by co-oxidative metabolism (11, 14). No known microorganisms exist capable of utilizing this compound as a sole source of carbon and energy (1, 14). Since cooxidative metabolism seldom leads to the incorporation of the carbon into bacterial cell mass, Alexander (1) has postulated that the persistence of compounds such as 2,4,5-T is due to the inability of natural microorganisms to derive their carbon and energy from the slow co-oxidative metabolism of this compound. We have recently developed a method, termed plasmidassisted molecular breeding, that has allowed us to breed in the laboratory a mixed culture capable of utilizing 2,4,5-T as a sole source of carbon and energy (10). In this article, we report on the isolation and properties of a pure culture capable of utilizing 2,4,5-T as a sole source of carbon and energy.
MATERIALS AND METHODS Organisms. Strain AC1100 was isolated after successive plating of the mixed culture from the chemostat 72
VOL. 44, 1982
BIODEGRADATION OF 2,4,5-T
73
0.8 U
00.6 10 ._
10.4 1=' 0.4
(. (U-
-
C._
6,t
IO
ONa-
m. co
0.2
0
1
2
3
4
5
6
Days FIG. 1. Growth of strain AC1100 with 2,4,5-T (1.5 mg/ml) as a sole source of carbon and Symbols: A, optical density; 0, viable count; 0, percent chloride release.
tion for each assay was determined by recording 02 uptake by the suspension for 3 to 7 min before the addition of substrates. Net 02 uptake was determined by subtracting the activity before substrate addition from that after substrate addition. Determination of chloride release. Chloride ion concentration in culture fluids was determined directly with a chloride ion-specific electrode (Lazar Research Labs, Inc.) that was standardized against a wide range of known chloride standards.
RESULTS
Isolation of a pure culture. We have previously demonstrated that the technique of plasmidassisted molecular breeding allows the development of a mixed culture that can utilize 2,4,5-T as a sole source of carbon and energy for its continued growth in a chemostat (10). Initially, plating of such a culture on nutrient agar plates demonstrated the presence of three or four types of colonies with titers of about 107 cells per ml in the chemostat, but plating on a minimal 2,4,5-T (BSM-2,4,5-T) plate would not allow any colony formation. Continued selection of such a mixed culture with 2,4,5-T as the sole carbon source in the chemostat and occasional plating on BSM2,4,5-T plates, however, later demonstrated the appearance of tiny colonies on such plates. Continued streaking of such colonies on BSM-
energy
in BSM.
2,4,5-T plates eventually produced colonies that could grow with 2,4,5-T as a sole carbon source with a generation time varying from 250 to 600 min, depending upon 2,4,5-T concentrations, media composition, and rate of aeration. Successive single-colony isolations from BSM2,4,5-T plates produced a pure culture, which has been tentatively identified as P. cepacia. This taxonomic identification is based on the fact that it is a strictly aerobic, pentose- and hexose-nonfermenting, gram-negative, and motile rod-shaped bacterium with multiple polar flagella. It is cytochrome oxidase and L-lysine decarboxylase positive, but L-arginine dihydrolase negative, grows at 30 and 37°C, but very poorly at 42°C, hydrolyzes Tween 80, and produces yellowish pigment in triple sugar iron agar. This culture has been designated as P. cepacia AC1100. Growth of AC1100 with 2,4,5-T. The ability of the strain AC1100 to grow with 2,4,5-T as a sole source of carbon and energy is shown in Fig. 1. There was a lag of almost 2 days before active growth ensued, as judged by an increase in the turbidity of the flask, as well as by an increase in the number of viable cells. To determine whether the growth was at the expense of total degradation of 2,4,5-T, we determined the extent of
74
APPL. ENVIRON. MICROBIOL.
KILBANE ET AL.
0.8
(96) i (97)
5
= 0.6
0
(49) (98)
4-'
_
=
4
0.4
C.,_
OC= u.z
3
2I
4
Days FIG. 2. Growth and chloride release by strain AC1100 during growth in BSM with different concentrations of 2,4,5-T. The numbers in parentheses indicate the percentage of chloride release in the medium after 5 days of growth. Symbols: 0, 500 ,ug of 2,4,5-T per ml; *, 1 mg of 2,4,5-T per ml; OI, 2 mg of 2,4,5-T per ml; A, 3 mg of 2,4,5-T per ml.
chloride release, as well as the physical loss of 2,4,5-T. At the end of the 6-day period, there was more than 97% loss of 2,4,5-T and essentially 100% release of chloride from the substrate. The effect of varying concentrations of 2,4,5-T on the growth and chloride release by AC1100 is seen from the results in Fig. 2. With increasing concentrations of 2,4,5-T, there was a decrease in the growth rate of the strain, and at concentrations above 2 mg of 2,4,5-T per ml in the medium, there was a pronounced lag before active growth resumed. Under such conditions, however, the ultimate cell density was higher than with lower concentrations of 2,4,5-T. No growth was observed when the 2,4,5-T concentration was increased beyond 3 mg/ml. The effect of varying temperature and pH on growth and 2,4,5-T degradation was also studied (Table 1). As shown in Table 1, maximum growth and 2,4,5-T degradation occurred at 30°C and pH 7.0. Although the culture could grow slowly at 20 and 37°C and at pH 6.0, there was
little growth when the pH of the medium was above 8.0 or the temperature above 40°C. Degradation of 2,4,5-T by resting cells of strain AC1100. The ability of P. cepacia AC1100 cells to utilize 2,4,5-T was further studied by incubating 2,4,5-T-grown washed cells of AC1100 (about 2 x 108 cells per ml) in 50 mM phosphate buffer (pH 7.0) containing 1 mM MgSO4 with 2,4,5-T as the only source of carbon. At zero time, as well as at different periods of incubation, samples were withdrawn, the cells were centrifuged, and the amount of 2,4,5-T in the supernatant liquid was determined by gas chromatography. The amount of chloride released from 2,4,5-T was measured by a chloride-selective electrode. On incubation with resting cells, the concentration of 2,4,5-T was progressively decreased, and by about 9 h, 90% of the 2,4,5-T was degraded by the resting cells. No residual 2,4,5-T could be detected at the end of the 24-h period. That the 2,4,5-T is completely degraded with the release of the three chlorines can be
BIODEGRADATION OF 2,4,5-T
VOL. 44, 1982 TABLE 1. Effect of temperature and pH on the degradation of 2,4,5-T by P. cepacia AC1100 2,4,5-T degradation measured by: pH Growtha Temp pH Growth'
(OC) 0 20 30 37 42
ClP release
+ +++ ++ -
7.0 7.0 7.0 7.0 7.0
(%)
Physical loss (%)b
