Physiological factors affecting carbon tetrachloride dehalogenation by ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1993,

0099-2240/93/051635-07$02.00/0 Copyright C) 1993, American Society for Microbiology

p. 1635-1641

Vol. 59, No. 5

Physiological Factors Affecting Carbon Tetrachloride Dehalogenation by the Denitrifying Bacterium Pseudomonas sp. Strain KCt R. L. CRAWFORD* Department of Bacteriology and Biochemistry and Center for Hazardous Waste Remediation Research, University of Idaho, Moscow, Idaho 83843 THOMAS A. LEWIS

AND

Received 11 December 1992/Accepted 11 March 1993

Pseudomonas sp. strain KC was grown on a medium with a low content of transition metals in order to examine the conditions for carbon tetrachloride (CT) transformation. Several carbon sources, including acetate, glucose, glycerol, and glutamate, were able to support CT transformation. The chelators 2,2'-dipyridyl and 1,10-phenanthroline stimulated CT transformation in a rich medium that otherwise did not support this activity. Low (< 10 ,uM) additions of dissolved iron(II), iron(III), and cobalt(II), as well as an insoluble iron(III) compound, ferric oxyhydroxide, inhibited CT transformation. The addition of 50 FM iron to actively growing cultures resulted in delayed inhibition of CT transformation. CT transformation was seen in aerobic cultures of KC, but with reduced efficiency compared with denitrifying cultures. Inhibition of CT transformation by iron was also seen in aerobically grown cultures. Optimal conditions were used in searching for effective CT transformation activity among denitrifying enrichments grown from samples of aquifer material. No activity comparable to that of Pseudomonas sp. strain KC was found among 16 samples tested.

Carbon tetrachloride (CT) is a carcinogenic, ozone-depleting, xenobiotic compound that also has acute liver toxicity in animals. Before these characteristics of CT were known, it was widely used as a solvent, industrial degreasing agent, fire extinguisher, and grain fumigant. CT has been found in groundwaters and is listed as a priority pollutant by the U.S. Environmental Protection Agency, mandating cleanup of contaminated sites. In aqueous solution, CT is not readily hydrolyzed and has an estimated half-life of 7,000 years (11). Because of CT's recalcitrance to spontaneous degradation, conditions favorable for dehalogenation must be created in order to effect remediation of CT contamination. Because the carbon atom of CT is fully halogenated, no net oxidation is possible; however, reduction is favorable and will proceed in the presence of a suitable reductant. Reductive transformations would be expected to proceed through a trichloromethyl radical formed after transfer of one electron to CT and release of a Cl atom as Cl-. The trichloromethyl radical may then accept an H atom to form CHCl3, another toxic pollutant which is less readily reduced. Alternatively, another electron may be transferred to the trichloromethyl radical to form dichlorocarbene, which could hydrolyze to form formate or CO (5). If 02 were present, combining a molecule of 02 with a trichloromethyl radical would lead to production of phosgene, which could also hydrolyze, forming CO2. The latter two possibilities are attractive from a remediation standpoint because they offer the potential for conversion to nonhalogenated products in rapid sequence. To initiate such a reaction, an appropriate reductant would be required. Although chemical reductants are available, microorganisms, with the addition of relatively inexpensive substrates, have greater potential for the cost-effective production of such reducing power. Microbial CT transformation processes that effectively

convert CT to nonhalogenated products have been described. These processes include those carried out by a number of strictly anaerobic bacterial cultures (2, 3, 8) and a denitrifying culture (1). Each of these may be useful for treating CT contamination, depending on how easily conditions favorable to each active population can be created. In this respect, the use of denitrifiers for the in situ treatment of oxygen-saturated oligotrophic groundwater seems advantageous because of their facultative anaerobic metabolism, use of a soluble electron acceptor, nonfastidious growth factor requirements and substrate range, and almost ubiquitous distribution in environmental samples. The many unknowns about the properties of denitrification-linked CT transformation make it difficult to assess, either positively or negatively, its utility for in situ applications. Criddle et al., after discovering a pure culture with effective CT transformation activity under denitrifying conditions (Pseudomonas sp. strain KC), noted that this activity was hampered by iron, cobalt, and possibly vanadium supplementation, and by oxygen (1). Activity was not observed in nongrowing cell suspensions. A requirement for copper has been noted more recently (10). In addition to those limitations, the prevalence of this type of CT-transforming activity may be rare, because only one effective enrichment was found among six samples of aquifer material. Here, we describe studies of CT transformation aimed at determining the potential of Pseudomonas sp. strain KC as a bioaugmentation strain, as well as a search for KC-type CT transformation activity in samples from regional aquifers.

MATERUILS AND METHODS Bacterial strains and culture conditions. Pseudomonas sp. strain KC was obtained from C. Criddle. Pseudomonas denitnficans (ATCC 13867) was obtained from the American Type Culture Collection. Medium D was prepared as described by Criddle et al. (1); the precipitate formed after autoclaving was removed by filtering the medium through a

* Corresponding author. t Publication no. 92523 of the Idaho Agricultural Experiment

Station.

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LEWIS AND CRAWFORD

0.45-,um (pore size) membrane, dispensing 100-ml portions into 160-ml serum bottles, and autoclaving again. HEPES (N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered minimal medium (HM) contained, per liter of deionized water, 11.9 g of HEPES (Research Organics, Inc., Cleveland, Ohio), 1 g of NH4SO4, 2 g of sodium acetate, 1 g of sodium nitrate, 1 ml of 1 M MgSO4, and 10 ml of 10 mM Ca(NO3) or, for nitrate-free media, CaCl2. The pH of this medium was adjusted to 7.6 to 7.9 with NaOH. Three milliliters of 1.5 M K2HPO4 and 50 ,u of 0.1 mM CuCl2 were added after autoclaving. The preparation of piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO) 2H20 sodium salt (Research Organics, Inc.)-buffered minimal medium (PM) was as described for HM, except that 19.8 g of POPSO replaced HEPES and pH was adjusted with HCl. Other carbon sources or nutrients used in minimal medium were glucose (American Chemical Society reagent; Sigma Chemical Co., St. Louis, Mo.), glycerol and ethylene glycol (Baker reagent; J. T. Baker Chemical Co., Phillipsburg, N.J.), glutamic acid (Sigma grade), ethanol (95%), and Casamino Acids (type R; Marcor Development Corp., Hackensack, N.J.). A solution of 10% iron-free Casamino Acids was prepared by extracting the original solution with 3% 8-hydroxyquinoline in chloroform and sparging with air for 1 h to remove residual chloroform before autoclaving. Amorphous iron oxyhydroxide was prepared as described by Lovley and Phillips (7), with two centrifugation and washing steps, and was diluted in deionized water to give a suspension of approximately 20 mM iron per liter before autoclaving. Nutrient broth or nutrient agar (Difco Laboratories, Detroit, Mich.) was used as rich medium and for routine maintenance of cultures. Anaerobic medium was purged of air by sparging with N2 through stainless steel cannulae in serum bottles for 5 min at a flow rate of 100 ml/min before being sealed with slotted grey butyl rubber stoppers and autoclaving. Inocula were grown aerobically, without iron supplementation unless otherwise noted, from single colonies by shaking in test tubes (16 by 150 mm) in a slant rack on a rotary shaker maintained at 30°C. Cultures were grown in 160-ml serum bottles or 28-ml Balch tubes. Growth was monitored turbidometrically by measuring A550 with a Hewlett-Packard 8652 diode array spectrophotometer or a Bausch & Lomb Spectronic 2000 for Balch tubes. Whole-cell protein was determined by the bicinchoninic acid method (Pierce Chemical Co., Rockford, Ill.) after solubilizing protein by heating washed cell pellets at 100°C for 20 min in 1 M NaOH. CT transformation in liquid cultures. For containment of volatiles, stoppers were replaced with sterile Teflon Mininert valves or Teflon-faced grey butyl rubber stoppers (West Co., Phoenixville, Penn.) after addition of volatiles. For denitrifying cultures, stoppers were replaced in an anaerobic chamber with an atmosphere of 85% N2, 10% H2, and 5% CO2. CT was added from a 6% (vol/vol) solution in methanol with a Hamilton 1702 gas-tight syringe. Cultures containing CT were incubated in an inverted position at 25°C. The headspace of aerobic cultures was flushed with 02 before stoppering and adding of CT. Dissolved oxygen in 2.0-ml samples was measured in a YSI 5301 stirred cell at 25°C by using a YSI 4004 dissolved oxygen probe (Yellow Springs Instrument Co., Yellow Springs, Ohio) calibrated with degassed N2-saturated deionized water, air-saturated deionized water, and O2-sparged deionized water as standards of 0, 8.6, and 41.3 mg of 02 per liter, respectively. Nitrate was measured with a Dionex series 4000 ion chromatograph

APPL. ENvIRON. MICROBIOL.

(Dionex Corp., Sunnyvale, Calif.) equipped with a conductivity detector. A Dionex AS5 column was used, with 2.2 mM Na2CO3, and 2.8 mM NaHCO3 as eluent at 1.5 ml/min. Sampling and inoculation were done with sterile 25-gauge stainless steel needles. Samples were removed with a Hamilton 1005LT gas-tight syringe. Quantitation of halomethanes. CT, CF (CHCl3), and MC (CH2Cl2) were quantitated by using headspace gas chromatography. Liquid samples were collected from vessels equilibrated to 25°C (ambient temperature) and placed in 10-mlheadspace autosampler vials and stoppered immediately with rubber stoppers and aluminum crimp seals. Vials were then placed in a Hewlett-Packard 19395 headspace autosampler with a constant-heating-time magazine. The sample carousel was heated to 70°C. At this temperature, a 5-min run time afforded a 15-min equilibration time for each vial. The injection sequence included a 10-s pressurization, followed 10 s later by a 1-s vent-sample loop fill, followed 1 s later by injection to the gas chromatograph. The auxiliary gas (helium) pressure was 1.4 x 105 Pa. The sample loop, a section of 0.02-in. ([0.05-cm] inner diameter) stainless steel tubing with a volume of 44 ,ul, was maintained at 75°C. Helium flow was maintained at 160 ml/min to a HewlettPackard 5890 gas chromatograph equipped with an electroncapture detector and DB-624 column (30 by 0.32 mm by 1.8 ,um; J&W Scientific, Inc., Folsom, Calif.). The column flow rate was 1.6 ml/min. Operating conditions for the gas chromatograph were as follows: injector temperature, 200°C; oven temperature, 100°C; and detector temperature, 300°C. The detector make-up gas was 90% argon and 10% methane. The detector signal was collected by an IBM PS2-55SX computer by using a Beckman 406 analog interface module and processing data with System Gold software (Beckman Instruments, Inc., Fullerton, Calif.). Calibration was performed by use of a multilevel set of standards prepared in 1 ml of culture medium in 10-ml-headspace autosampler vials stoppered with rubber stoppers, to which CT, CF, and MC from stock solutions were added. Stock solutions of CT, CF, and MC were prepared in methanol by adding the individual components to a 10-ml volumetric flask containing methanol and weighing it between additions on a Sartorius R200D analytical balance. CT removal was calculated as CT added to culture minus CT remaining at the end of the experiment. Total CT was calculated from the liquid CT concentration by using a dimensionless Henry's law constant of 1.244 (4). Determinations of Henry's constant for CT in HM were within error of this value. 14C radiotracer studies. 14C-labeled CT (4.3 mCi/mmol; New England Nuclear, Boston, Mass.) was added to culture vessels from a methanol solution to give approximately 60,000 dpm per culture. Unlabeled CT was also added to give a final specific activity of 0.25 mCi/mmol. Triplicate cultures that included 14C-labeled CT were stoppered with rubber stoppers and incubated in an inverted position. At the end of the experiment, 14C was accounted for in volatile and liquid fractions as follows. Volatiles were purged from the culture vessels by connecting the bottles to a trapping train consisting of three scintillation vials filled with 10 ml of toluene scintillation cocktail followed by one vial filled with 10 ml of Carbosorb C02-trapping solution. Purging was accomplished by inserting a 6-in. (15-cm) 16-gauge stainless steel cannula connected to an N2 line into the liquid phase and inserting an 18-gauge syringe needle connected to the trapping train just below the stopper. The gas stream was then bubbled through at a flow rate of 100 to 150 ml/min. After the bottles were purged for 30 min, the gas lines and

VOL. 59, 1993

DEHALOGENATION OF CT BY PSEUDOMONAS SP. STRAIN KC

1637

TABLE 1. Percentage recovery of '4C in radiotracer experiments % Recovery of 14C

Conditions

NonoltiecCell Nonvolatile' 2 associatedd

Volatile

organiec Sterile, 14C-CT Sterile, 14C-HC03-

KC, N2, 14c-CT KC, 02, 14c-CT

87.3 0.2 9.9 36.9

+ 14.4 + 0.01 ± 5.4 ± 19.8

7.5 95.5 21.4 32.0

± 2.9 ± 3.8 ± 5.2

+ 18.9

1.0 + 0.6 ND 62.3 ± 2.0 6.5 + 2.1

NDf ND

5.5 ± 2.3 4.8 + 2.1

Ttl Total'

95.8 95.5 99.1 80.0

± ± ± ±

11.1

3.9 11.1 8.5

a Mean disintegrations per minute trapped in toluene plus mean disintegrations per minute lost in second purge after alkalinizing with NaOH + sum standard deviation (SD) (triplicate experiments). b Mean disintegrations per minute trapped in Carbosorb plus mean disintegrations per minute lost in second purge after acidifying with HCI + sum SD. c Mean (+ SD) disintegrations per minute remaining in liquid sample after acidifying and purging. d Mean (+ SD) disintegrations per minute lost by filtration through a 0.2-p.m (pore size) membrane. e Mean (+ SD) disintegrations per minute trapped in toluene plus disintegrations per minute trapped in Carbosorb plus disintegrations per minute in liquid sample before second purge. f ND, not determined.

stopper were removed, and the two toluene vials were counted directly in a Beckman LS7000 scintillation counter. One milliliter of the Carbosorb was added to 10 ml of Bio-safe scintillation cocktail (RPI, Mt. Prospect, Ill.) and counted. Liquid samples were removed and processed as follows. A 1-ml sample of liquid was added to a vial containing 10 ml of scintillation cocktail and counted. A second 1-ml sample was added to a vial containing 0.1 ml of 40% NaOH; N2 was bubbled through for 1 min at 500 ml/min before scintillation cocktail was added, and the vial was counted. A third 1-ml sample was added to a vial containing 0.085 ml of concentrated HCI; N2 was bubbled through for 1 min before scintillation cocktail was added, and the vial was counted. A fourth sample was filtered through a 0.2-,um (pore size) membrane (Gelman Acrodisc, Ann Arbor, Mich.) and added to a vial containing 0.085 ml of HCG; N2 was bubbled through before scintillation cocktail was added, and the vial was counted. To reduce chemiluminescence, vials containing NaOH or Carbosorb were left for 2 to 3 h before they were counted. Counts per minute were converted to disintegrations per minute by using H numbers and an algorithm obtained from counting a set of quenched standards. The counts obtained from the first liquid sample were used as the total counts in the liquid fraction; the counts in the second liquid sample were used to represent total liquid counts minus volatiles not stripped by the initial purge; the counts in the third liquid sample were used to represent total liquid counts minus volatiles and CO2, and the counts in the fourth vial were used to represent total liquid counts minus volatiles, CO2, and cell-associated (bound and sorbed) radioactivity (Table 1). Enrichment for CT transformation activity. Aquifer solids were obtained from test wells drilled at two locations: one on the Hanford Nuclear Reservation in south-central Washington and another at a Superfund site in southern Idaho. Samples from the Hanford site were obtained by sterile sampling techniques that were able to trace possible contamination from drilling fluids (6). The Hanford samples were packaged under argon and shipped on ice by overnight carrier for processing the next day. Samples from the southern Idaho site were obtained by split-spoon sampling with no special measures taken to prevent or detect surface contamination. These samples were also shipped on ice by overnight carrier and processed within 48 h. Enrichments were carried out by adding several grams of the wet aquifer material to 100 ml of sterile, anaerobic medium D, which was

then incubated at 25°C for 1 week. Inocula were transferred from the initial enrichments to fresh medium D containing CT to reduce potential interferences from aquifer materials. Material from the southern Idaho site (RW3-79') was used in seeding experiments with Pseudomonas sp. strain KC. Seeding was accomplished by washing a stationary-phase culture of strain KC in a sterile solution (pH 7.0) containing 11 mM NaCl, 0.6 mM Mg(H2PO4)2, 0.35 mM KCG, and 0.05 mM CaCl2. The washed suspension was diluted in the same solution, and 4 ml of each dilution was added to 40-g samples of aquifer solids and stored in glass jars with Teflon-lined screw-cap lids held at 4GC. RESULTS Medium for study of denitrification-linked CT transformation. The characteristics of the medium in which the CTtransforming activity of KC was discovered were low content of iron and cobalt and a trace amount of copper (1, 10). These requirements were further characterized by investigating the growth of KC and transformation of CT in HM, which was formulated to avoid a metal precipitation step while retaining a low-metal composition. The growth of KC in HM was dependent on an additional organic carbon source and a nitrogen source. Batch cultures maintained a pH within 0.2 pH unit of the initial value at stationary phase; the optimal growth of KC took place at pH values between 7.5 and 7.9. The growth of KC was not flocculent on HM under denitrifying conditions or under aerobic conditions when iron supplementation was included. Flocculence was observed in aerobic, iron-starved cultures and with potassium concentrations of

0

15

0.2

W,fl

E 0)

0.1

5

U

/

0.0O

5

0

45

10 40 MM Metal Added

0

FIG. 4. Effect of added Fe(II), Fe(III), and Co(II) on CT removal by KC cultures. *, Fe(II)C12; 0, Fe(III)C13; V, amorphous iron(III) oxyhydroxide; El, CoCl2.

24

36 48 Time (hours)

60

72

36 48 Time (hours)

60

72

1600 is

1400 F

1200j

IN 1000 1-

800 _

0.40 0

600 _

0.35 400 _

0.30 _ 200 E

0.25 _

0r in

0

to

0.10 _ 0.05

96

72

Time (hrs.) 2500

1

2000

1500

1 000

500

0

0

24

FIG. 6. Effect of iron supplementation during iron-limited growth upon CT transformation by KC. 0, no iron supplementation; *, 50 F.M FeSO4; *, 50 ,uM FeC13; V, 200 ,uM amorphous iron(III) oxyhydroxide; V, 1 mM sodium azide. Arrows indicate time of supplementation. O.D., optical density.

0.15 _

0

./

0

0.20 _

12

24

36

48

Time (hrs.)

FIG. 5. Effect of iron status of KC inoculum on CT transformation. Solid symbols, 50 p.M FeSO4; open symbols, no iron supplementation. Ol and *, inoculum grown with 50 ,uM FeSO4; 0 and *, inoculum grown without iron supplementation; ----, calculated loss due to sampling. Abs., absorbance.

the first day, each culture responded to growth at low levels of iron by an increased rate of CT transformation and responded to growth at high levels of iron by a loss of this activity. The addition of 50 ,uM FeSO4, FeC13, or approximately 200 ,uM amorphous iron(III) oxyhydroxide to exponentially growing, iron-starved KC resulted in a delayed decrease in CT transformation rate relative to the controls, whereas the addition of azide resulted in an immediate cessation of CT removal (Fig. 6). Effect of oxygen on CT transformation. The effect of oxygen on CT removal by KC was studied in order to model the performance of KC in an air-saturated aquifer. In this experiment, an excess Of 02 was provided to one culture after removing the initial liquid samples (4 ml) by replacement with an excess of 02 (6.5 ml) through a sterile 0.2-,um (pore size) filter. Thereafter, liquid sample volumes were replaced with an equal volume of 02. The other cultures received air or N2. CT transformation was seen in all cultures, although it was seen to a greater extent in the cultures with low dissolved oxygen content (Fig. 7), even though the yield of cells was much greater in the aerobic culture (24.3 + 0.4 and 61.8 + 3.9 p.g of protein per ml, respectively). In a separate experiment, no significant NO3 consumption was seen in a culture of KC grown with an 02

1640

._~

APPL. ENVIRON. MICROBIOL.

LEWIS AND CRAWFORD 800 700 600 500 400 300 200 1 00

0

0

24

48

1 .4 1.2 1.0

Er 0

0.8

LO

5 0.6 16 0.4 0.2 0.01

72

Time (hrs.)

Time (hrs.) 30 25

0 E

20

U~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ a LUz o

0

>5

o 15 0

_

5

t

0

24

48

INz F-

72

Time (hrs.)

FIG. 7. CT transformation by aerobic versus anaerobic KC. O, sterile, 02-flushed headspace; *, KC 02-flushed headspace; 0, KC air headspace; *, KC anaerobic headspace; --- --, CF production anaerobic headspace; CF production air headspace.

0

24

48 Time (hrs.)

72

FIG. 8. Effect of iron on CT transformation by aerobic KC cultures. *, no iron supplementation; V, 2.75 ,uM FeSO4; El, 27.5 pAM FeSO4. O.D., optical density.

--,

headspace under the same conditions (not shown). CF production was greater in the anaerobic culture than in the cultures grown with 02. As in anaerobic cultures, the addition of iron decreased CT removal by KC under aerobic conditions (Fig. 8). The product spectra of the aerobic and anaerobic transformation processes were measured by using 14C-labelled CT. The distribution of radioactivity in the aerobic KC culture differed significantly from that in the anaerobic culture. A larger fraction remained as volatile organictrapped material, as expected from the reduction in transformation efficiency seen previously. In addition, a larger fraction was trapped as C02-acid volatile material, and a significant amount (20%) was not recovered. Prevalence of hydrolytic CT transformation in denitrifying enrichments. Samples of material from aquifers in southcentral Washington and southern Idaho were enriched for denitrifying bacteria as described by Criddle et al. (1). The resulting cultures were transferred to fresh media containing CT to test for the presence of CT transformation activity minimizing contaminants from aquifer materials. No cultures were found with the activity of Pseudomonas sp. strain KC, which showed an effective removal of CT with little CF production (not shown). The survival of Pseudomonas sp.

strain KC in aquifer materials stored at 4°C for 1 year and its ability to express CT transformation activity after enrichment in microcosms were confirmed by use of dilutions of stationary-phase KC added to aquifer material. The amount of activity seen in the samples was dependent on the initial spike of KC and the medium used to test the enrichment (Fig. 9). Six other samples were retested in HM with or without spiked addition of 104 CFU of stationary-phase KC per g of aquifer material. As in the previous experiment, these samples were enriched on medium D before 1 ml of the resulting enrichment was transferred to 100 ml HM. Only spiked samples showed effective CT removal (data not shown). DISCUSSION The extent to which studies with defined laboratory cultures can be used to predict the efficacy of a biological remediation process depends upon what is learned of the underlying physiology of the remediation process. We have characterized several variables affecting CT transformation by a denitrifying pseudomonad. Because the dehalogenation catalysts of Pseudomonas sp. strain KC are not known, it is unclear how directly these factors affect CT transformation. Biochemical effectors, which can alter the flow of electrons to an active reductant, competitively inhibit CT reduction, or influence the synthesis of an active reductant, would affect

VOL. 59, 1993

DEHALOGENATION OF CT BY PSEUDOMONAS SP. STRAIN KC Medium D

although not as completely as iron supplementation does. This indicates that the catalyst is not completely susceptible to oxidation by 02 in preference to CT, nor is it produced exclusively under conditions of anaerobic iron limitation. It is also interesting that CT transformation in the presence of 02 is a "cleaner" process, yielding less CF and more CO2. This may result from competition for trichloromethyl radicals between 02 and other reacting species. Regardless of what the catalyst produced by KC may be, the reductive hydrolysis of CT is a relatively rare occurrence among organisms enriched under denitrifying conditions (1; this study). There is certainly potential for the use of KC for CT remediation. Its sensitivity toward metal ions may seriously limit the success of in situ processes unless site characteristics include a very low availability of metal. Genetic improvement may offer a solution, should it be possible to derive constitutive variants. For aboveground processes, metal removal may allow maximal activity by KC. Because oxygen did not prevent CT transformation by KC, there is potential for in situ activity even in air-saturated environments or possibly in vapor treatment systems.

1400

= 1200 0

1000 0

L

800 600

o

1641

400

E 200 0

PM-acetate 1 600

1400

ACKNOWLEDGMENTS This work was supported by Battelle Pacific Northwest Laboratories, Richland, Washington, under subcontract 097710-A-L2. We thank J. Fredrickson and T. Stevens for access to DOE subsurface samples, C. Criddle for the gift of Pseudomonas sp. strain KC, K. Stormo and L. Deobald for expert technical advice, and C. Bollinger for editorial assistance.

c 1200 o

1000

0

800 O

600

o

400

E

s O)

200

1.~ I E

0

1

2

3

4

5

6

7

8

FIG. 9. Detection of CT transformation activity in aquifer solids seeded with KC and stored at 4'C for 1 year. Open bars represent CT removed after 7 days; solid bars represent CF produced in 7 days. Inocula are from the following enrichment cultures on medium D: 1, none; 2, unseeded control enrichment; 3, sample seeded with 2 x 104 CFU of KC per g; 4, sample seeded with 2 x 105 CFU of KC per g; 5, sample seeded with 2 x 106 CFU of KC per g; 6, sample seeded with 2 x 107 CFU of KC per g; and 7, pure culture of KC.

reductive transformation of CT. The iron status of a bacterial cell is a major determinant of its redox biochemistry because many of the proteins involved in electron transport contain iron. The addition of ferrous or ferric iron to iron-limited cultures of KC resulted in delayed inhibition of CT transformation. Because the forms of iron used were in the molar excess of CT, it appears that they do not compete more successfully for electrons than CT. Because Fe(II) chelators stimulated CT transformation and Co(II) inhibited CT transformation, Fe(II) is implicated as the effector of iron inhibition. In addition, because cobalt supplementation would not lead to the synthesis of iron-containing redox proteins, a diversion of electron flow away from CT transformation by these proteins would not explain inhibition by cobalt. The fact that copper is required for maximal CT transformation suggests that it plays a role in the synthesis of the active component, either as an integral part or in a biosynthetic role. Oxygen would be expected to compete with CT for electrons because of its high standard redox potential. High 02 concentrations do reduce CT transformation activity,

REFERENCES 1. Criddle, C. S., J. T. DeWitt, D. Grbic-Galic, and P. L. McCarty. 1990. Transformation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions. Appl. Environ. Microbiol. 56:3240-3246. 2. Egli, C., T. Tschan, R. Scholtz, A. M. Cook, and T. Leisinger. 1988. Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobactenum woodii. Appl. Environ. Microbiol. 54:2819-2824. 3. Gauli, R., and P. L. McCarty. 1989. Biotransformation of 1,1,1-trichloroethane, trichloromethane, and tetrachloromethane by a Clostridium sp. Appl. Environ. Microbiol. 55:837-844. 4. Gossett, J. M. 1987. Measurement of Henry's Law constants for C1 and C2 chlorinated hydrocarbons. Environ. Sci. Technol. 21:202-208. 5. Krone, U. E., R. K. Thauer, H. P. C. Hogenkamp, and K. Steinbach. 1991. Reductive formation of carbon monoxide from CCl4 and FREONs 11, 12, and 13 catalyzed by corrinoids. Biochemistry 30:2713-2719. 6. Long, P. E., J. P. McKinley, T. J. Phelps, S. A. Rawson, and F. S. Colwell. Unpublished data. 7. Lovley, D. R., and C. J. P. Phillips. 1986. Organic matter mineralization with reduction of ferric iron in anaerobic sediments. Appl. Environ. Microbiol. 51:683-689. 8. Mikesell, M. D., and S. A. Boyd. 1990. Dechlorination of chloroform by Methanosarcina strains. Appl. Environ. Microbiol. 56:1198-1201. 9. Stumm, W., and J. J. Morgan. 1981. Aquatic chemistry: an introduction emphasizing chemical equilibria in natural waters, 2nd ed., p. 456. John Wiley & Sons, New York. 10. Tatara, G., B. Fathepure, A. Rhoades, and C. Criddle. 1992. Novel aspects of the transformation of carbon tetrachloride by Pseudomonas sp. strain KC, abstr. Q-286, p. 383. Abstr. 92nd Gen. Meet. Am. Soc. Microbiol. 1992. American Society for Microbiology, Washington, D.C. 11. Vogel, T. M., C. S. Criddle, and P. L. McCarty. 1987. Transformations of halogenated aliphatic compounds. Environ. Sci. Technol. 21:722-736.