Environ. Sci. Techno/. 1995,29, 2856-2863
Biotranh-on
of Carbon
S Y E D A . HASHSHAM,+ R I C H A R D SCHOLZE,* A N D DAVID L. FREEDMAN*,' Environmental Engineering and Science Program, Department of Civil Engineering, University of Illinois, 205 North Mathews Avenue, Urbana, Illinois 61801, and U.S. Army Construction Engineering Research Laboratory, Champaign, Illinois 61820
Biotransformation of carbon tetrachloride (CT) was examined with an anaerobic enrichment culture grown on dichloromethane as the sole organic carbon and energy source. The principal products from [14C]CT included chloroform (17%), carbon disulfide (21%), and COS (21%). When cyanocobalamin was added along with CT, the percentage converted to COS increased almost 3-fold (59%), while CS2 decreased somewhat (1l%), and virtually none of the CT (-=lo/,) was reduced to chloroform. Carbon monoxide was a major transformation product (12-27%) in autoclaved cultures and in live cultures that received high levels of CT (up to 52 mg/L). Adding cyanocobalamin also increased the rate of CT transformation in live cultures by a t least 10-fold, but had a minor effect on the rate of CT use in autoclaved cultures. Accelerated rates of transformation by live cultures were sustained for as long as 200 days, with hydrogen serving as the electron donor. Cyanocobalamin, hydroxocobalamin, and methylcobalamin were equally effective, while a 3-week lag period was required before adenosylcobalamin started to enhance CT transformation. Because of their high cost, the feasibility of using cobalamins will depend on the amount required. W e observed significant enhancement in CT transformation at concentrations up to 340pM, with cobalamin levels as low as 10 pM.
Introduction Carbon tetrachloride (CT) has been one of the most widely used chlorinated solvents over the past six decades, with annual U.S.production levels reaching as high as 525 million kg. Substantial amounts of CT have been released to the environment, resulting in stratospheric ozone depletion (1, 2) and groundwater contamination (3). Because of its carcinogenicand toxic properties, the maximum contami* Corresponding authortelephone: (217)333-9017; fax: (217)3339464; e-mail address: freedman@w:l.cso.uiuc.edu. +
University of Illinois. U.S. Army Construction Engineering Research Laboratory.
2856 1 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 11, 1995
nant level for CT in drinking water has been set at 5 pg/L (4). Pump-and-treat remediation of groundwater contaminated with CT and other dense, nonaqueous phase liquids has met with limited success (5). In situ bioremediation is a promising alternative, but its application to compounds such as CT has thus far been limited. This is due in part to the accumulation of hazardous metabolites and the inhibition of biotransformation when CT is present in the milligram per liter range. Numerous studies on microbial transformation of CT under anaerobic conditions indicate that competing pathways are involved. Reductive dechlorination of CT to chloroform (CF) and dichloromethane (DCM) occurs readily. Further reduction to chloromethane and methane has been reported, but the rate and extent do not appear to be useful from aremediation standpoint (6, 7). Reductive dechlorination of CT is therefore undesirable if anaerobic conditions alone are used during remediation. Reduction of CT to carbon monoxide and/or formate also occurs, and both of these compounds can subsequently be oxidized to COZ. This pathway is more desirable in terms of yielding nonhazardous products. The abiotic reduction of CT with iron porphyrins (8-121, factor F430 (7, 101, and corrinoids (6, 7,10,13-15) in the presence of an electron donor is also known to yield a mixture of chlorinated and nonchlorinated products. These results demonstrate the significant contribution of nonenzymatic reactions to the transformation of CT in the presence of live organisms. Several recent studies have also demonstrated abiotic conversion of CT to carbon disulfide in the presence of sulfides (16, 17).As with the formation of CF and DCM, the formation of CS:! from CT is undesirable given its well-known neurotoxic properties. The initial concentration of CT appears to influence which pathway predominates. Many microbial studieshave been carried out at CT levels below 1 mg/L (18-22). However, the concentration in contaminated groundwaters is often higher (3,231,especially if free-phase CT is present. Above 1 mg/L, reductive dechlorination is typically significant, leading to the accumulation of CF and DCM (15, 19, 24-26). None of the studies that have examined biotransformation of CT in the mglLrange under acetogenic or methanogenic conditions have reported more than 26% net hydrolysis to COz. Becker and Freedman (27) demonstrated the potential to enhance CF biodegradation by adding low levels of cyanocobalamin (CN-Cbl) to a mixed methanogenic enrichment culture grown on DCM. The addition of CN-Cbl increased the rate of CF biodegradation approximately 10fold, increased the extent of CF oxidation to Cop, and minimized the accumulation of DCM and chloromethane. In this study, we examined the possibility of enhancing CT biotransformation in the same culture using several cobalamin homologues, with similarly favorable results. The addition of cobalamins increased the rate of CT transformation, increased the fraction of CT converted to COZ,and decreased the formation of CF. Carbon monoxide, CS2, and several water-soluble compounds were also identified as transformation products.
0013-936XJ95/0929-2856$09.00/0
G 1995 American Chemical Society
Materials and Methods Chemicals. CT, CF, and DCM (299.9%)were obtainedfrom Aldrich Chemical Co. CT was added to cultures as a CTsaturated basal medium solution (approximately6.8 mM) for amounts below 14 pmol or as neat liquid for higher levels. CS2 was purchased from EM Industries, Inc. Cobalamins (98+%,Sigma Chemical Co.) were added from 2 mM stock solutions and stored at 4 "C in foil covered bottles. CO and chloromethane (CM; 99.5%, Matheson), methane (99%;Matheson), C02 (anaerobic grade; Linde), a 30% C02-70% N2 mixture (Linde),and H2 were obtained from Liquid Air Corporation. [l4C1CT was purchased from Dupont NEN Products and diluted to 6.04 x lo6 disintegrations per minute (dprn) per mL with distilled deionized water. Scinti-Verse-E(FisherScientific)was used as liquid scintillation cocktail. Enrichment Culture. The anaerobic enrichmentculture used in this studywas developed by Freedman and Gossett (28). DCM serves as the sole organic carbon and electron donor source, oxidizing a portion to COZ plus H2 and fermenting the balance to acetate. Methanogens convert the acetate and C02 plus HZ to CHI. Procedures for maintenance of the stock culture are described elsewhere (27). In this study, the culture was grown in two media, which differed only in their iron content: 29 mg1L Fe2+in medium I and 1.2 mg/L Fe2+ in medium 11. All other components in the media are described elsewhere (29). The lower iron level in medium I1 resulted in much less iron sulfide precipitate, making it possible to more easily monitor growth on DCM. CT transformationstudies were initiated by transferring (in an anaerobic glovebox; Coy Laboratory Products, Inc.) 100mL of the DCM-degrading enrichment culture (without dilution) to 160-mL serum bottles. The stock culture was brought back to its original volume with basal medium. All bottles were then purged with 30% C02-70% NZ gas and sealed with slotted gray butyl rubber septa and aluminum crimp caps. One addition of DCM (typically23pmol/bottle) was made prior to adding CT to ensure the culture retained its DCM degrading capability after the transfer. The serum bottles were incubated at 35 "C, in the dark, on a shaker table, in an inverted position (so that the liquid remained in contact with the septum). Bottles that received cobalamins were covered with aluminum foil to prevent photolysis. Hydrogen was periodically added to some bottles as an electron donor, using a 1-mL Pressure-Lok gas-tight syringe (Dynatech Precision Sampling Corp.). Water controls consisted of 100 mL of deionized water plus CT. Methane controls consisted of 100 mL of stock enrichmentculture to which no CT or cabalaminwasadded, in order to assess the endogenousamount of electron donor available from the stock culture. Analpis of Volatile Compounds. CT, CF, DCM, CM, CHI, and CSZwere measured by gas chromatographic(GC; Perkin Elmer 9000)/flameionization analysis of a 0.5-mL sample taken from the 60-mL headspace of the serum bottles, as previously described (28,29). Although flame ionization is not an ideal quantification method for sulfur compounds like CS2, a linear detector response was demonstrated for up to 50 pmol per serum bottle. In this study, we report the total mass of each compound present in a 160-mL serum bottle. The percentage of each compound in the 100-mLaqueous phase was 4.8%for CH4, 3.4% for CO, 77% for CM, 93% for DCM, 88% for CF, 47%
for CT, and 56%for CS2. These values were calculated using Henry's constants [ ( m ~ l a m gas - ~ concentration) 1( m ~ l - m - ~ aqueous concentration)] at 35 "C reported previously (293 4 , except for CS2. Its Henry's constant at 35 "C is 1.22, which was determined according to Freedman et al. (32). HZwas measured using a Perkin-Elmer GC (Model9000) equipped with a thermal conductivity detector and a 3.2 mm x 3.2 m stainless steel, 1001120mesh Carbosieve S-I1 column (Supelco,Inc.). The column was run isothermally at 60 "C with nitrogen as the carrier gas (30 mL/min). CO was analyzed on a GC (GOW-MACInstruments) equipped with a 3.2 mm x 2.44 m stainless steel 80/100 mesh, Molecular Sieve 5A column and thermal conductivity detector. The column was operated isothermally at 50 "C with helium carrier gas (30 mL1min). 14C-labeledvolatile compounds were analyzed by a GC combustion technique described previously (29). Headspace samples were separated on the appropriate GC column; as each compound eluted, it was oxidized to 14C02 in a quartz tube filled with CuO (800"C),trapped in NaOH, and counted in scintillation cocktail. The total dpm in a bottle for each compound was calculated based on its Henry's constant at 35 "C. Presumptive confirmation of 14C0was achieved by coelution of authentic material on two different columns, as previously described (27). Confirmation of 14CS2was obtained by coelution of authentic material on two columns (1%SP-1000Carbopack B and GP 10%columns; Supelco, Inc.) and by GC (Hewlett Packard Model 5890A)/mass spectrometric (Fisons Inc., Model 70VSE) analysis with a DB-5 capillary column (15 m). 14C02 and 14C-LabeledNonvolatile Compounds.
14C02
and 14C-labelednonvolatile compounds were measured as previously described (28) after completing analysis of the 14C-labeledvolatile compounds. Once NaOH was added to the serum bottles (driving all 14C02 into the liquid), aliquots were removed, acidified, and purged with NZ through an NaOH trap; the 14C recovered in NaOH corresponded to 14C02; the 14C not purged at acid pH corresponded to nonstrippable residue (NSR). The nonfilterable (retained by a 0.45-pm filter) fraction of 14C-NSR activity was presumed to be cell associated material. The compositionof the 14C-labeledNSR filtrate (soluble fraction) was determined by high-performance liquid chromatography, as described previously (28). Aqueous sampleswere acidified and sparged to remove carbonates, then filtered (0.45pm), and injected onto a 300-mm HPX87H Aminex ion exclusion column (Bio-RadLaboratories) connected to a UV/vis absorbance detector (Model 486, Millipore Corp.). As compoundseluted, theywere collected with a fraction collector and counted. The column was run at two different temperatures (30 and 65 "C) to aid in the separation of several closely eluting compounds. Presumptive identification of formate, acetate, methanol, and butyrate was made according to coelution with authentic material at both column temperatures. The average percent recovery for soluble NSR analysis was 88% (one standard deviation = 4%). The overall efficiency of the 14C analysis method was defined as the total dpm recovered in all components (CT CF DCM CM CH4 CS2 CO CO2 NSR) divided by the total dpm present in a serum bottle at the time of 14Canalysis. The denominator was determined by summing the dpm in the headspace (0.5 mL of sample added to scintillation cocktail)plus liquid (1OOpLof sample
+
+
+
+
+
+
+
+
VOL. 29, NO. 11, 1995 /ENVIRONMENTAL SCIENCE &TECHNOLOGY
2857
2'oP -
a
['oo
T
...... a CS? 0
X .
0
*.O
.
.
.
I
.
io
5
.
.
.
. . . . . .
,
15
20
1
0.0
0
b ............. .....................
...........
.....
0
DCM
io
5
20
15
E
cs2 ..... .......0 -......3 .......0 .......... o..o ...........o..* .......0.......
,
0 2.0
5
.
;
.
:
10
7
:
15
.
;
.
3
20
* d ....... ............
...........
....... 0....' . Q
1.o
0.0 0
5
10
15
20
Time (days)
FIGURE 1. CT transformation (0)and CS2 (0).CF (A), and DCM (0) formation in live cultures with CN-CbI added (a); live cultures with no CN-CbI added (b); autoclaved cultures with CN-CbI added (c); and autoclaved cultures with no CN-CbI added (d). The average of duplicate bottles is shown.
added directly to scintillation cocktail). On average, the overall I4C recovery was 109% (one standard deviation = 6%).
Results [l4C1CTTransformation with CN-Cbl. These experiments were conducted with the DCM-degrading enrichment culture grown in medium I. All bottles received an initial CT addition of 2 pmol plus 7.5 x lo5 dpm of [I4C]CT.As shown in Figure 1, the consumption of CT was at least 10 times faster in the live cultures supplemented with CN-Cbl (0.2pmollbottle), without significant accumulation of CF or DCM. The addition of CN-Cbl to autoclaved cultures improved the initial rate of CT transformation, but the total time required was approximately the same as without the supplement. CF increased and then declined in both sets of autoclaved cultures. Table 1 shows the distribution of 14C recovered from [14C]CT. The principal products in CN-Cbl supplemented live cultures were C02,soluble NSR, and CSz. Without CNCbl added, the principal products were CF, COz, soluble NSR, and CS2. The soluble NSR compounds included (presumptively)methanol, formate, acetate, and butyrate. Autoclaving resulted in a significant amount of 14C0 formation,with more than twice as much recovered in the bottles that received CN-Cbl. Approximately 20% of the CT was transformed to COz in autoclaved cultures with or without the addition of CN-Cbl, essentiallythe same as in 2858 # ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 11. 1995
the live cultures that did not receive CN-Cbl. [14C]CFwas present in only trace amounts in both sets of autoclaved cultures. The nonfilterable amount of 14Cwas highest in the autoclaved bottles and lowest in the live cultures that were supplemented with CN-Cbl. In general, CT was inhibitoryto methanogenesis. During this experiment, however, methane output rose slightly in the live bottles that received CT and CN-Cbl (Figure la). The relatively low level of CT added and its rapid rate of transformation may have combined to allow for some methanogenesis. A low but reproducible percentage of the transformed [l4C1CT was recovered as radiolabeled methane (1.9%),while no 14CH4was detected in any of the other bottles (Table 1). The addition of CT to the live bottles completely inhibited methanogenesis, so methane levels are not shown in Figure lb. Long-Term CT Transformation. The effectiveness of CN-Cblwas also tested in eight serum bottles that were set up in the same manner as the [l4C1CTbottles, startingwith the DCM enrichment culture grown in medium I. However, rather than one dose of CT, these bottles received repeated additions for as long as 200 days. As shown in Figure 2, the supplemental addition of CN-Cbl significantly increased the rate of CT transformation. A total of 4.8 pmol of CNCbl was added over the first 48 days. The average rate of CT transformation during this interval was 0.95 pmol bottle-' day-', nearly seven times higher than in the bottles that received only CT. During 200 days of incubation, the loss of CT from duplicate water controls was minor (1.5 pmollbottle) compared to the total amounts of CT consumed in the live and autoclaved bottles (Table 2). With time, the rate of CT use began to slow down in the bottles that did and did not receive CN-Cbl (Figure 2). In an effort to restore the initial rate of CT transformation in the cobalamin-amended bottles, more CN-Cbl (totaling4.4 pmol) was added between days 82 and 105, but without a noticeable effect. Within the same interval, CH4output in the methane control bottles began to level off, signaling a depletion of endogenous electron donors that were present when the bottles were set up. More electron donor was therefore added to all the bottles that received CT, in the form of hydrogen, with a subsequent improvement in the rate of CT use. A total of 220pmol of HZwas added between days 109 and 200. Although H2 increased the rate of CT use in both sets of live bottles, the effect was more pronounced in the ones that received CN-Cbl. The addition of CT to the live cultures completely inhibited methanogenesis, even with CN-Cbl added. By comparison, the average CI-& output from duplicate control bottles (no CT or CN-Cbl added) was 83 pmol during the 200 days of incubation. This reflected the endogenous amount of electron donor available to the cultures when they were set up. CT transformation in autoclaved bottles was also examined for an extended period, although the ones receiving CN-Cblwere operated for only 48 days. As shown in Table 2, the average rate of CT transformation in the autoclaved bottles during the first 48 days of operation was similar to the rate for the live cultures that did not receive CN-Cbl(O.11-0.14pmol bottle-'day-'). AdditionofHzto the autoclaved bottles did not improve the rate of CT transformation, while it resulted in a noticeable improvement in the live cultures. Table 2 shows the net amount of CF, DCM, and CS2 formed as a percentage of the CT consumed during long-
TABLE 1
Distribution of
14C
(96 dpm) Recovered from Transformation of [14C]CTB
component CT CF DCM
csz CH4
co
live culture, no CN-CbI
live culture, CN-CbI added
autoclaved culture, no CN-CLI
autoclaved culture, CN-CbI added
0.84 ( f 0 . 1 6 ) 17 ( f 1 . 9 ) 1.1 (3~0.43) 21 ( f 1 . 9 ) 0
0.48 ( f 0 . 0 7 ) 0.53 ( f 0 . 7 5 ) 0 11 ( f 0 . 1 5 ) 1.9 (f0.13) 0 59 ( f 1 . 9 ) 4.2 ( f 1 . 3 ) 3.7 ( f 1 . 6 ) 6.1 ( f 2 . 6 ) 3.2 ( f 0 . 7 1 ) 4.3 ( f 0 . 9 3 ) 5.3 ( f 0 . 5 4 )
0.99 (f0.22) 0.58 (f0.011 1.6 ( f 0 . 4 9 ) 19 ( f 0 . 3 7 ) 0 12 ( f 2 . 2 ) 21 ( f 0 . 9 4 ) 6.2 ( f 0 . 7 5 ) 6.8 ( f l . 1 ) 11 ( f 0 . 7 7 ) 4.1 (f0.05) 8.0 ( f 0 . 6 5 ) 9.4 ( f 0 . 3 9 )
0.76 ( f 0 . 3 0 ) 0 0 16 ( f 0 . 6 7 )
0 21 ( f 0 . 1 7 ) 6.4 (rt0.25) 4.7 (4~0.70) 6.9 ( f 0 . 3 3 ) 6.2 ( f 0 . 5 3 ) 5.9 ( f 0 . 2 1 ) 9.4 (*0.20)
c02
acetateb formateb methanolb butyratelisobutyrateb other soluble NSR nonfilterable NSR
0 27 (f3.3) 17 (fO.lO) 4.9 ( f 0 . 4 3 ) 4.7 (*0.69) 11 ( f 0 . 1 3 ) 4.6 ( f 0 . 6 7 ) 6.2 (rtl.6) 8.1 (3ZO.19)
a Values in parentheses represent one standard deviation for duplicate bottles. Presumptive identification based on coelution with authentic material. Butyrate and isobutyrate were not resolved.
No CN-Cbl Added
10
4
+
CN-Cbl Added
10
5
0 0
25
50
75
100 125
150
175 200
Time (days) FIGURE 2. Long-term CT transformation in live cultures with and without supplementaladdition of CN-CbI. Resultsfor duplicate bottles were very similar. Each arrow corresponds to a 20 pmol addition of Hz.
term incubation of the bottles. The highest percentage of CT reductively dechlorinated to CF occurred in the live cultures that did not receive CN-Cbl, the same as in the [l4C1CTexperiment. The live bottles supplemented with CN-Cbl transformed almost five times more CT during 200 days of operation, yet less than 1%accumulated as CF plus DCM. There was also very little accumulation of CF and DCM in both sets of autoclaved bottles. In a l l eight bottles, CSZlevels were consistently higher than CF or DCM (CO was not assayed during this experiment). Approximately two-thirds of the CT consumed in the live cultures supplemented with CN-Cblwas transformed to CS2 during the first 48 days. However, the level of CS2 in these bottles actually declined during the subsequent 152 days and
accounted for only 13% of the total CT consumed at the end of 200 days. CT TransformationwithCobalaminHomologues. This experiment compared the effect of CN-Cbl, hydroxocobalamin (OH-Cbl),methylcobalamin (MeCbl),and adenosylcobalamin (AdoCbl) on CT transformation, using the live enrichment culture grown in medium 11. Successive additions of increasing amounts of CT were made. Cobalamins were added every 3-4 days until a total of 1pmol was reached on day 15. As shown in Figure 3, the addition of OH-Cbl substantially increased the rate of CT consumption. The highest level of CT added was 72 pmol, resulting in an aqueous phase concentration of 52 mg/L. At this point, the molar ratio of OH-Cbl to CT added was 1.4%.CS2 was initially the principal volatile product, but its level declined after day 18. CO accumulated to a significant degree in these bottles. Figure 4 compares the cumulative amount of CT transformed in all of the homologue-amendedbottles. With the CN-Cbl, OH-Cbl, and MeCbl bottles, the rate of CT transformationbegan to slow by day 24. HZadditions (on days 34 and 401 restored the CT consumptionrate, although most of the H2 was not consumed; out of 120pmol added, an average of 88pmolremained on day 64. Methanogenesis was completely inhibited in all of the bottles, regardless of cobalamin addition. With AdoCbl, a 24-day lag was experienced before it began to accelerate the rate of CT transformation. By day 40, these boples nearly caughtup with the other cobalaminamended bottles. Table 3 summarizesthe total amount of CT transformed for each pair of bottles as well as the volatile products formed according to GC analysis. In all of the cobalamin-amended bottles, less than 1.6% of the CT consumed was reductively dechlorinated to CF plus DCM, compared to 18%in the unamended bottles. As with OHCbl, CS2 in the CN-Cbl, MeCbl, and AdoCbl bottles rose initially and then declined,while CO increased steadily and became the major volatile product.
Discussion The results of this study demonstrated that cobalamin homologues enhance anaerobictransformationof CT, even at CT concentrations as high as 52 mg/L. With cobalamin addition, the rate of CT transformation increased at least 10-fold. At the same time, cobalamins added to live cultures VOL. 29, NO. 11, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
2859
TABLE 2
Long=Term Transformation of CTa live cultures NOCN-CbI day 0-48 day 49-200
av rate of CT
CN-CbI day 49-200
NO CN-CbI day 49-200
day 0-48
CN-Cbl day 0-48
0.14 (10.001) 0.20 (f0.001) 0.95 (h0.056) 0.89 (f0.017) 0.11 (f0.005) 0.12 (fO.009) 0.14 (f0.036)
transformation (pnol bottle-' cumulative CT transformed (pmollbottle) net CF formed (% of CT transformed) net DCM formed (% of CT transformed) net CS?formed (% of CT transformed) CN-Cbl added (pmollbottle) a
day 0-48
autoclaved cultures
6.7 (*0.03)
31 ( f 0 . 2 )
46 ( f 2 . 7 )
134 (12.6)
5.3 ( f 0 . 2 2 )
19 ( 1 1 . 3 )
6.5 ( f 1 . 7 )
9.9 (f0.55)
5.6 (f0.75)
0.7 (h0.02)
0.4 ( f 0 . 0 3 )
3.7 (10.84)
1.0 (fO.O1)
4.6 (12.7)
1.3 (h0.06)
0.1 (50.07) 0.5 (h0.07)
0.2 (10.01)
0.9 ( f 0 . 1 5 )
0
0.3 (f0.08)
29 kt3.4)
26 kk0.3)
67 ( f 0 . 2 )
Oc
25 ( f 5 . 9 )
9 (f3.9)
42 ( f 2 . 9 )
0
0
4.8
4.4
0
0
4.8
Average of duplicate bottles; numbers in parentheses represent one standard deviation. CO was not measured. Calculated as the total amount
of CT consumed divided by the number of days per interval. OThe amount of CS? present in both bottles actually declined in this interval.
.
120 1
a OH-Cbl added
\ ............._
0..
0
20
40
60
80
0
20
40
60
Time (days) FIGURE 4. Cumulative transformation of CT in live enrichment cultures supplemented with CN-CbI (0).OH-CbI (A), MeCbl (O), AdoCbl (01,and no Cbl (W).
0
20
40
60
80
Time (days) FIGURE 3. CT transformation in bottles with and without OH-Cbl added (a) and formation of CO (01,CF (A). and CSZ 1 . 4 in the bottle supplemented with OH-CbI (b). Results for duplicate bottles were very similar. Each arrow corresponds to a 40 p n o l addition of Hz. The principal volatile productsmeasured in the CT only bottles (not shown) ware CSt and CF.
caused a shift in metabolite distribution, away from reductive dechlorinationand toward net hydrolysis. During the [l4C1CTexperiments, nearly three times more CT was converted to C02 in live culturesthat received CN-Cbl (Table 1). The initial aqueous phase concentration of CT in these tests was approximately 1.5 mg/L. Only Bouwer and McCarty (18)have reported a higher percent net hydrolysis of CT to COein a biotic system, although they worked with comparatively low levels of CT (20-50 pglL). With most anaerobic pure cultures, including Acetobacterium woodii and Methanobacteriumthermoautotrophicum,the percent 2860
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 1 1 , 1 9 9 5
conversion of CT to COZhas typically been reported in the range of 10-15% (15,25,26).Under denitnfyingconditions, Pseudomonas strain KC converts approximately one-half of the CT it consumes to COz (33). The conversion of CT to COZ appears to proceed by several pathways. One is the reduction of CT to CO and/or formate,which can subsequently be oxidized to C02. Only lowlevels of '*C-labeled formate were detected (
