Effects of Medium and Trace Metals on Kinetics of Carbon

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0099-2240/93/072126-06$02.00/0

Effects

of Medium and Trace Metals on Kinetics

of Carbon

Tetrachloride

Transformation

by

Pseudomonas

sp.

Strain KC

GREGORY M.TATARA,"12 MICHAEL J. DYBAS,2 AND CRAIG S. CRIDDLE2,3*

Departmentof Microbiologyand PublicHealth,1Departmentof Civiland EnvironmentalEngineering,3and National Science Foundation CenterforMicrobialEcology,2MichiganState University,

East Lansing, Michigan 48824 Received 19 January1993/Accepted29April1993

Underdenitrifying conditions, Pseudomonas sp. strain KCtransforms carbontetrachloride(CT)tocarbon dioxide via acomplexbut asyet undetermined mechanism.Transformationrates werefirst order withrespect toCT concentration over the CT concentration range examined (0to100,ug/liter) andproportionaltoprotein concentration, giving pseudo-second-order kineticsoverall.Additionof ferric iron(1 to20,uM) to an

actively

transforming culture inhibited CTtransformation, and the degree of inhibition increasedwithincreasingiron concentration. By removing iron fromthe trace metals solution orby removing iron-containing precipitate from the growth medium, higher second-orderratecoefficientswereobtained. Copperalsoplaysarole in CT transformation.Copper was toxic at neutral pH. Byadjusting the medium pHto8.2,solubleiron and copper levels decreased as aprecipitate formed, and CT transformationratesincreased.However,culturesgrownat high pH without any added trace copper(1,uM) exhibited slower growthratesand

greatly

reducedratesof CT transformation, indicating that copper is required for CT transformation. The use of pH adjustment to decrease iron

solubility,

to avoid copper

toxicity,

and to provide a selective advantage for strain KC was evaluatedby using soil slurriesandgroundwatercontaining highlevelsof iron. Insamples adjustedtopH8.2 and inoculated with strain KC, CT disappeared rapidly in the absence or presence ofacetate or nitrate supplements. CTdid notdisappearinpH-adjustedcontrols that were not inoculatedwithstrain KC.

In recent years, considerable interest has surrounded prospects for degrading hazardous contaminants in situby stimulating selected bacterial populations(biostimulation)or byaddition of novel organisms to contaminated sites

(bio-augmentation). Stimulation of an indigenous population is

likely to yield an enrichment that is well adapted to its

environment, whereas foreign organisms introduced into

suchanenvironment may be unabletocompete. However, introduced organisms do offer certain advantages provided thattheycan compete with the indigenous microflora. Un-like indigenous organisms, introduced organisms can be

extensively studied and understood in the laboratory,

im-proving prospects for control of their activity in the field. Amongother reasons, control of activity is needed to avoid

theproductionof unwanted by-products. Chloroform (CF),

forexample, is a commonendproduct of carbon tetrachlo-ride (CT) transformation in both laboratory and field envi-ronments(2,4, 5,7). CF is more persistent than CT in many

environments, andit is also a suspected carcinogen.

Conse-quently,metabolicpathways that do not produce CF are of

interest. Pseudomonas sp. strain KC is an aquifer-derived organism that transforms CT to CO2and unidentified non-volatile products without CF production under denitrifying conditions (3). Related Pseudomonas species grown and assayed under the sameconditions do not transform CT.

Here we report on the kinetics of CT transformation by Pseudomonas sp. strain KC, and we describe experiments to evaluate the role of trace metals in CT transformation kinetics. Finally, we provide evidence that accelerated CT transformation can be obtained in iron-rich groundwaters andsoil slurries by adding strain KC after pH adjustment.

* Correspondingauthor.

MATERIALSANDMETHODS

Chemicals. CT (99% purity) was obtained from Aldrich Chemical Co., Milwaukee, Wis. All chemicalsfor prepara-tion of media were ACS reagent grade (Aldrich or Sigma Chemical Co.),and allwaterusedwas18-Mohmresistance orgreater.

Preparation of media and growth conditions.Medium D(3) contained(perliter of deionizedwater)2.0 gofKYH2PO4,3.5 gofK2HPO4, 1.0 g of(NH4)2SO4,0.5 gofMgSO4 7H20,1 ml oftracenutrientstock TN2, 1 ml of 0.15 MCa(NO3)2,3.0 gofsodium acetate, and 2.0 g of sodium nitrate. Medium D wasprepared withtracenutrient stock solution TN2. Stock solution TN2contained (perliter of deionizedwater) 1.36 g of

FeSO4.

7H20, 0.24 g of

Na2MoO4.

2H20, 0.25 g of

CuSO4.

5H20, 0.58 g of

ZnSO4.

7H20, 0.29 g of

Co(NO3)2.

6H20, 0.11 g of NiSO4- 6H20, 35 mg of Na2SeO3, 62 mg of H3B03, 0.12 g of NH4V03, 1.01 g of

MnSO4*

H20, and 1 ml ofH2SO4 (concentrated). Insome

experiments,differenttracemetalpreparations were usedto

study their effects on CT transformation. TN2-Cu and

TN2-Fe stock solutions lacked CUSO4 5H20 and

FeSO4.

7H20,respectively, butwereotherwise identicalto TN2. After addition of all essential medium components, mediumD wasadjustedto adesired initial pH of 8.0 or 8.2 with 3 NKOH. This finaladjustment in pH resulted in the formation ofawhiteprecipitate. The resulting mediumwas autoclaved at 121°C for 30 min and transferredto an anaer-obicglove box for degassing.

Precipitate-free medium Dwas prepared as follows.

Me-dium D (adjusted to an initial pH of 8.0 or 8.2) was

autoclaved at 121°C for 30 min, transferredto an anaerobic glove box for degassing and quiescent settling of precipitate, and decanted after 24 h. The precipitate-free and oxygen-free decanted mediumwasreautoclaved for30 minat121°C and cooled beforeuse. Precipitate-free medium Dcontained

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CT TRANSFORMATION BY PSEUDOMONAS SP. STRAIN KC 2127

24 mM acetate, 25 mM P043-, 19 mM NO3-, and 3.8 nM iron, as determined by atomic absorption spectroscopy and ionchromatography.

Cultures were grown under an N2 atmosphere in one of three different containers: (i) 28-ml serum tubes (Bellco Glass no.2048-00150), (ii) a modified 1-liter Wheaton bottle asdescribed by Balch and Wolfe (1), and (iii) 250-ml (8-oz) bottles sealed with screw-cap Mininert valves (Alltech cat-alog no. 95326). Both the serum tubes and the modified Wheaton bottles were sealed with Teflon-faced butyl rubber septa (West catalog no. 1014-4852) and aluminum crimp seals. All cultures were shaken at 100 to 150 rpm at 20 to 23°C. Strain KC did nottransformCT at temperatures above 25°C, and it did not grow at temperatures above 30°C (data notshown). Culturemanipulations were typically performed in a Coy anaerobic glove box (Coy Laboratories, Ann Arbor, Mich.) under an atmosphere of 98% N2 and 2%H2. Oxygen level wasmonitored continuously with a Coy model 10 gas detector. Hungate technique was used for anaerobic manip-ulations outside the glove box.

Analytical methods. All bottles used to evaluate CT trans-formation were sealed with pressure tested screw-cap Mininert valves or Teflon-lined butyl rubber stoppers. CT was assayed by removing 0.1 ml of headspace gas with a 0.25- or0.5-ml Precision gastight syringe (Alltech catalog no. 050032), closing the syringe valve, inserting the syringe needle through the gas chromatograph (GC) injection port septum, opening the syringe valve, and injecting the sample into the GC. For parts-per-billion concentrations, the GC was a Perkin Elmer model 8500 equipped with a 100/120-mesh column (10% Alltech CS-10 on Chromsorb W-AW; Alltech catalog no. 12009 PC) and an electron capture detector with nitrogen carrier (40 ml/min) and nitrogen makeup (27 ml/min). For parts-per-million concentrations, the GC was a Hewlett Packard model 5890 operated isother-mally at 150°C and equipped with a DG 624 column (J&W

Scientificcatalog no. 125-1334) and a flame ionization

detec-tor (hydrogen flow rate = 100 ml/min; air flow rate = 250 ml/min). The carrier gas was nitrogen (16

ml/min).

External standard calibration curves were prepared by

addition of a primary standard (7.8 ng of CT per ,lI of

methanol or 0.82 ,ug of CT per,ul of methanol) to secondary standard water solutions having the same gas/water ratio, ionic strength, incubation temperature, and speed of shaking as the assay samples. A four-point calibration curve was prepared over a concentration range bracketing that of the assaysamples. Protein was stored by freezing at -20°C and

assayedby the modified Lowry method, with bovine serum

albumin as the standard (6).

Masstransfer rates were estimated for 28-ml serum tubes containing 10 ml of medium D and sealed with Teflon-lined rubber septa. CT was added to the aqueous phase, and the tubes were shaken at 100 rpm on a shaker table. Headspace CT was assayed every 60 s for 10 min by GC. The mass transfer ratekLa (per hour) was determined by plotting ln [1

-

(Cg/Cg)]

versus shaking time, where Cg is headspace CT concentration and Cg is headspace CT concentration at

equilibrium.Theslope of the resulting plot is -kLa[(Vg/Vaq)

+

(1IH,)],

where

H,

is the dimensionless Henry's constant (1.0 for CT at 20°C), Vaq is water volume, and

Vg

is headspace volume.

Determination of reaction rate order and transformation

capacity. The dependence of CT transformation rate on CT

concentration was assessed with stationary-phase cultures grownfor 72 h froma 1% inoculum in both medium D and

precipitate-free medium D. Cultures were dispensed into

28-ml serum tubes, and CT (5 to 100

jig/liter)

was added. The tubes were then transferredto a shaker table, and headspace CT was periodically monitored by GC. Reaction rates were calculated from measurements taken after 20 min had elapsed to allow sufficient time for equilibration of head-space CT with the water-phase CT. Under these conditions, the mass transfer rate (-25 h-1) was much greater than the reaction rate (-1 h-1). The observed rates were corrected for equilibrium partitioning into the gas phase to obtain the true reaction rates (see below). To evaluate the dependence of CT transformation rate on total culture protein, a station-ary-phase culture was diluted 1:5, 1:3, and 1:2 with medium and then monitored for CT removal by sampling of the gas phase.

To determine the transformation capacity of KC cultures grown under different conditions, 100 ml of stationary-phase culture (grown for 72 h in 1-liter modified Wheaton bottles in either medium D or precipitate-free medium D) was dis-pensed into 170-ml serum vials sealed with Teflon-lined septa. The vials were then spiked with CT to give an initial concentration of 1 or 5 mg/liter, shaken at 100 rpm at

20°C,

andmonitored for CT removal by sampling of the gas phase.

Effects of trace metals. To assess the effects of trace

copper, medium D was prepared with either stock solution TN2 or TN2-Cu, transferred to 8-oz (250-ml) bottles, sealed, autoclaved, cooled, and inoculated with a 1% inoc-ulum of a stationary-phase culture of Pseudomonas sp. strain KC. Cultures were grown to stationary phase, spiked withCT, and assayed for CT transformation.

To assess the effects of trace iron, medium D and precip-itate-free medium D were prepared by using trace metal stock solutions TN2 and TN2-Fe. Cultures were grown for 48 or 72 h, spiked withCT, and assayed for CT transforma-tion. To assess iron inhibition, 10 ml of early-stationary-phase culture (grown for 72 h in precipitate-free medium D) wastransferred to 28-ml serum tubes in an anaerobic glove box, spiked with 0 to 20,uM ferric iron (as ferric ammonium sulfate), and equilibrated for 10 min. The serum tubes were sealed with Teflon-lined rubber stoppers, spiked with CT, shaken throughout the experiment on a shaker table, and monitored by sampling of the gas phase.

Transformation in groundwater and soil systems. The groundwater used in bioaugmentation experiments was Michigan State University tap water containing 0.051 mg of Feper liter. After the pH of the groundwater was adjusted to 8.2 with 3 N KOH, unsterilized groundwater or filter-sterilized (0.22-pum-pore-size filter) groundwater was dis-pensed into a suite of autoclaved 120-ml serum bottles. Some bottles served as uninoculated controls for abiotic losses. Theremainder were inoculated with a 1% inoculum of strain KC grown on 1% nutrient broth (Difco Co.). Some of the inoculated bottles were autoclaved, while others received additions of acetate (300 mg/liter as sodium acetate) and nitrate (200 mg/liter as sodium nitrate). The headspace above all samples was replaced with nitrogen, but no effort was made toremove oxygen dissolved in the water. All bottles were sealed withTeflon-lined rubber stoppers, spiked with 1.5 ,ug ofCT, placed on a shaker table, and monitored by sampling of the gas phase.

Soil slurryexperiments were conducted by using Metea-type soil from the B horizon at Michigan State University (0.7% organic matter, 31 ppm of iron, 4.8 ppm of nitrate, and 9.9 ppm ofammonia). Soil slurries (286 g in 100 ml of tap water)adjusted to pH 8.2 with 3 N KOH were dispensed into 120-ml serum vials. Some samples were sealed and auto-claved to serve as abiotic controls for sorption and volatil-VOL.59,1993

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ization losses. Controls for the possible transformation of

CTbyindigenousmicroflorawereprepared bysealingserum

bottles with orwithout the addition ofacetate (300 mg/liter as sodium acetate) and nitrate (200 mg/liter as sodium

nitrate). The remaining bottles received a 1% inoculum of

strain KC(grown onprecipitate-freemediumD), giving an initial cell density of 5 x 102 cells per ml. Some of the inoculated bottles were amended with acetate (300mg/liter as sodium acetate) and nitrate (200 mg/liter as sodium nitrate). The headspace above allsampleswasreplaced with nitrogen, but no effort was made to remove dissolved oxygen. All samples were sealed with Teflon-lined rubber stoppers,spiked with 1.5 ,ug ofCT, placedon ashakertable, and monitoredbysamplingof the gasphase.

Modeling. Separateexperiments (see Results)established that transformation of CTwasfirst order with respecttoCT concentration over the concentration range examined and first order with respect to total protein concentration. As-suming a second-order kinetic expression, the following mass balance can be written for a closed batch system in which avolatile aqueous-phase substrate is in equilibrium with its gasphase:

-

dMcr

k'

dt

kCaqXVaq

Vaq + HcVg q

(1) where

MCT

is the total mass of CT in the system

(milli-grams), or Caq(Vaq + HCVT), t is time (days), k' is the

second-order ratecoefficient (literspermilligramofprotein

per day), Caq is the aqueous-phase concentration of CT

(milligrams perliter), andXis the concentration ofprotein

(milligramsperliter). Separationof variables andintegration

ofequation 1 yields

In_

ln 1- =

M\T

Forareaction that is first order withrespectto CT

concen-tration, aplotof thelogarithmof themassof CT in the bottle

versustime shouldgiveastraightlinewithslopeof-k'XVaq/

(Vaq + HcVg).

RESULTS

Kinetics of CT transformation. The dependence of CT transformationrateson CTconcentrationwasevaluatedby

plotting the logarithm ofmassofCTversustime. As shown

inFig. 1,thisplotwaslinear,and theslopeswereessentially constant overtheconcentration rangeexamined, indicating

that the reaction could be represented as first order with

respect to CT concentration.

Toassess the dependence ofthe CT transformation rate on proteinconcentration, first-order rate coefficients of the culturewereplotted againsttotalsolution protein

concentra-tion. As shown in Fig. 2, rates were linearly related to

protein concentration range evaluated (6.25 to 22 ,ug/ml). Thus, apseudo-second-order rate expression (firstorder in CTconcentration and pseudo-first order in protein

concen-tration) was considered appropriate. Pseudo-second-order

rate coefficients were calculated from the slopes of the

logarithmofmass versus time plot.

Effects of trace metals. Removal of the precipitate that formed during preparation of medium D at pH 8.0 had a

profoundeffect on the iron level of the medium. As

deter-mined by atomic adsorption spectroscopy, precipitate-free

0--1 In(Cr mass) -2. y = -0.22179-7.5852e-3x RA2=0.990 y =-0.67244-9.401le-3x RA2=0. y = -1.4877-9.5731e-3x R 20 40 60 Time (min) 100ug/L 50ug/L 25 ug/L 80 100

FIG. 1. Logarithmof mass of CTremainingversus time. Error barsrepresentingthestandard deviations offourindependent sam-plesaregenerally less thanthedimensions of symbols used.

mediumDcontained 3.8nM iron. Removal of iron from the growthmedium resulted inimportanteffectsongrowthrate, proteinlevels, CTtransformation rate, and CT transforma-tion capacity. As shown inFig. 3, protein concentrationat the end of the growth phasewasgreaterfor cells grown in medium D(proteinconcentration=350,ug/ml)than for cells grown in medium D prepared with TN2-Fe (protein con-centration = 51 ,ug/ml), precipitate-free medium D(protein concentration = 46 ,ug/ml), and precipitate-free medium D prepared with TN2-Fe (protein concentration = 22.5 ,ug/ ml). These observations indicate that diminishedgrowthwas due to the removal ofiron. Cells grown in precipitate-free medium D hadhigher pseudo-second-order ratecoefficients

(Table1)butlower 24-h transformationcapacities(Fig.4and

5) compared with medium D. Pseudo-second-order rate

coefficientswerelowest in thehigh-ironmedia andhighestin the low-iron media (prepared with TN2-Fe or without precipitate).

As shown in Table 1, pseudo-second-order rate coeffi-cients forCT transformationgenerallydecreasedascultures aged from 48 to 72 h, indicating decay of transformation

activityascells entered thestationaryphase. Theexception

was cultures grown in medium D with TN2-Fe. These culturescontinuedtogrowbetween 48 and 72 hand showed

0.2

20//

0.1 0. L 0 10 20 30 Proteinconcentration

(gg/mL)

FIG. 2. Dependenceoffirst-orderrate coefficient on totalprotein concentration.

kXVaq

- t

Va

q +

Hc

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CT TRANSFORMATION BY PSEUDOMONAS SP. STRAIN KC 2129 .1 OD 660 0.9 0.6

.01

r.w

.001 I I 0 20 40 60 80 100 Time(hours)

FIG. 3. Growth ofPseudomonassp. strain KC in medium D and precipitate(Ppt)-free mediumD in the presence or absence of added iron. OD 600, opticaldensityat600 nm.

Cf (mg) 0.3 0.0 5 mg/LCr control 5mg/L Cr +KC 1mg/LCT control 1mg/LCr +KC 0 300 600 900 1200 1500 Time(min)

FIG. 4. EvaluationofCTtransformation capacity for mediumD. Errorbarsrepresentstandarddeviationsontriplicate samples.

no decrease in the second-order rate coefficient over this

period. Growthratesfor these cultures were higher and less

variable than those of cultures grown in precipitate-free media (Fig. 3). These observations suggest that for this

medium, cell growth and production of CT transformation

activitymay be controlled by thesolubilization of

contami-nantiron inthe precipitate.

The effect of addition of ferric iron to precipitate-free

mediumD-grown cellsisshown inFig.6. Addition offerric

ammonium sulfate (1, 5, 10, and 20

F.M)

to an actively

transforming stationary-phase culture inhibited the rate of

CTtransformation, and the degree of inhibition increasedas

theconcentrationofiron increased.

Criddleetal.(3)found that 1,uM copperprevented growth

ofPseudomonas sp. strain KC at neutral pH. Thepresent

work confirmed this finding. In medium D adjusted to pH

8.0, however, the maximum specific growth rate of strain

KCdecreased in the absence of copper,droppingfrom 0.047

h-1

forTN2+Cu to 0.016

h-1

forTN2-Cu. Final protein

concentration was not greatly affected by the presence or

absence ofcopper (185

Iag/ml

forTN2+Cu; 173 ,ug/ml for

TN2-Cu). As showninFig. 7, rapid

transformation

of CT

wasobtained only withTN2+Cu, andlittle orno

transfor-mation of CT was obtained withTN2-Cu.Thus, omissionof only 1 ,uMcopper was sufficienttoprevent CT transforma-tion.

Transformation ingroundwater and soil systems. As shown

TABLE 1. Effectsof ironlimitation and cultureageon second-orderratecoefficients for CT transformationby

Pseudomonassp. strainKC

Medium Culture k' (liters/mg of

modification_modification age_age(h) protein/day

±~~~~~-

1SD)

+ Precipitate 48 0.893 ±0.03 72 0.362 ±0.08 + Precipitate, - traceFe 48 3.93 ± 1.48 72 4.03 ±0.79 - Precipitate 48 6.18 ±0.48 72 2.28 ±0.45 - Precipitate, - traceFe 48 9.07± 1.24 72 4.41 + 0.56

in Fig. 8 and 9, inoculation ofgroundwateror soilslurries (pH adjusted to 8.2) with Pseudomonas sp. strain KC

increased the rate of CTtransformation. CT did not

disap-pear inpH-adjusted controls that were not inoculatedwith

strain KC. Addition of strain KC byitself was a sufficient

condition for CT transformation. Acetate and/or nitrate

additionswere notrequired.

DISCUSSION

Ourresults indicatethattransformationofCTby

Pseudo-monas sp. strain KC proceeds by a complex mechanism.

Thetransformation appearstobe linked to the

iron-scaveng-ingfunctions of thecell,aspreviously proposed(3). Obser-vationssupporting this hypothesis includethefollowing: (i) KCgrown inprecipitate-freemedium D doesnottransform CT if thegrowth medium is supplemented withtrace iron

before inoculationwith strainKC

(3),

(ii) additionof ironto

grown cultures of strain KC inhibits CT transformation,

possibly bycompetingfora

binding

site

(Fig. 6),

and

(iii)

the

second-order

rate coefficients for CT transformation

in-0.9 -0.6 -cr (mg) 0.3 -0.0 mi4,9T~~~~~~ 5mg/LCr control 5mg/LCT +KC 1mg/L

Cf

control 1mg/lCf +KC 0 300 600 9so 1200 1500 Time (min)

FIG. 5. Evaluation of CT transformation capacity for

precipi-tate-free medium D. Error barsrepresent standard deviations on

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0.20 Control 1 20PiMIron 0.15-10

pM

Iron 0.10 0.05-1jiMIron 0 jiMIron 0.00 II 0 50 100O 150 200 Time(min)

FIG. 6. InhibitionofCT transformationby variedconcentrations offerriciron. Error bars represent standard deviationson triplicate samples.

crease for cells grown in iron-limited media (Table 1).

Transformation of CTapparentlyrequirescopperand

prob-ably involves areducingagent, asevidencedbythe quench-ing action of oxidants, such as hydrogen peroxide and oxygen (datanotshown).

Given thecomplex mechanismpostulatedfor CT transfor-mation, it is likely that a complete kinetic description of the transformation will prove equally complex. The agent of transformation mustbe identified and quantified. More in-formation is needed tounderstand changes in CT transfor-mation activity with cell growth stage and trace metal species. As shown in Table 1, growing cells transform CT faster than dostationary-phase cells. There is also evidence that the CT transformation is affectedby other trace metals, notably cobalt and vanadium(3),and that thesetracemetals

act synergistically with iron to inhibit CT transformation

(la). In spite of these complexities, however, the present

results do establish a simple first-order dependence on CT

concentrationoverthe CT concentration range investigated.

Apseudo-first-order relationship with total protein

concen-0.8

--Copper

C(r

.g)

trationwasalso observed.However,useof totalproteinina

pseudo-second-order kineticexpressionmustbeviewedasa

temporary expediency. Totalprotein merelyfunctions as a

quantifiablesurrogatefor theactual agent oftransformation

until such time asthe agent itselfcanbe

quantified.

The presentwork presentsafavorable outlook for appli-cation of Pseudomonas sp. strain KC in field environments if thepH isproperlycontrolled. Adjustment of thepHof the groundwater and soil slurries to 8.2 was performed to decreaseironsolubility (3, 9), toavoid coppertoxicity, and

to provide a selective advantage for strain KC. In these

samples, CTwasremoved by the simple addition of strain KC. Acetate and nitrate additions were not required. An-other factorfavoringpossible applicationof strain KCis the

magnitudeof the second-order rate coefficients reported in

Table 1. These rates are highenoughfor reasonableratesof

transformation in an engineered in situ system. The

trans-formation capacity of strain KC also appears to be high

enough(>15 ,ugof CT per mg ofprotein) toenable removal

of CTatconcentrations that exceed thosereportedformost

Co-contaminated

sites (8). As shown inFig.4 and5, strain

KC removed up to 2.5 mg of

Co

per liter at a protein

0 5 10 15 20 25 30 0 2 4 6 8

Time(Hours)

FIG. 7. Effect of trace levels of copper on CT transformation Time (days) activity. Error bars represent standard deviations on triplicate FIG. 8. Transformation of CT in gre

samples. 8.2. Error barsrepresent standard deviat

)undwater adjusted to pH tionsontriplicate samples.

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CT TRANSFORMATION BY PSEUDOMONAS SP. STRAIN KC 2131

Cr 1 Autoclaved

(tug)

\ \ ;KConly

\I* 5 I Pr-sterilized soil+

\ . L vlbKC+acetate+nitrate

KC+acetate+nitrate

0 2 4 6 8

Trme(days)

FIG. 9. Transformation of CT in soilslurries adjustedtopH 8.2. Error barsrepresentstandard deviationsontriplicate samples.

concentration of 165,ug/ml (approximately 107cellsperml).

Finally, strain KC is a natural aquifer isolate capable of

growth and transformation of CToverthetemperaturerange

of5to 25°C,adesirablerange formostfield applications.

ACKNOWLEDGMENT

This work was supported by the NSF Center for Microbial Ecology at Michigan State University under National Science FoundationgrantBIR-9120006.

REFERENCES

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la.Criddle, C. S. 1989. Ph.D. thesis. Stanford University, Stanford, Calif.

2. Crddle,C. S., J. T. DeWitt, and P. L.McCarty.1990. Reductive dehalogenation of carbon tetrachloride byEscherichia coli K-12. Appl.Environ. Microbiol. 56:3247-3254.

3. Criddle, C. S., J. T. DeWitt, D.Grbic-Gali,and P. L.McCarty. 1990. Transformation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions. Appl. Environ. Microbiol.56:3240-3246.

4. Egli, C., R. Scholtz, A. M. Cook, and T. Leisinger. 1987. Anaerobic dechlorinationoftetrachloromethane and 1,2-dichlo-romethanetodegradableproducts by pure cultures of Desulfo-bacterium sp.andMethanobacterium sp. FEMS Microbiol.Lett. 43:257-261.

5. Egli,C.,T. Tschan, R. Scholtz, A. M. Cook, and T. Leisinger. 1988.Transformation oftetrachloromethane to dichloromethane andcarbondioxidebyAcetobactenium woodii. Appl. Environ. Microbiol. 54:2819-2823.

6. Markwell,M. A., S. M.Haas, N. E.Tolbert,and L. L.Bieber. 1981. Protein determination in membrane lipoprotein samples: manual andautomated procedures. Methods Enzymol. 72:296-301.

7. Semprini,L.,G.D.Hopkins,P.L.McCarty,and P. V. Roberts. 1992. In-situ transformation ofcarbon tetrachloride and other halogenated compounds resulting from biostimulationunder an-oxicconditions. Environ. Sci. Technol. 26:2454-2461.

8. Sittig,M.(ed.). 1985. Handbook of toxic and hazardous chemi-cals andcarcinogens, 2nd ed. Noyes Publications,NewYork. 9. Stumm,W., and J. J. Morgan. 1981.Aquatic chemistry,2nd ed.

John Wiley & Sons,NewYork. VOL. 59,1993

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