Environ. Sci. Techno/. 1995,29,762-768
Methanethiol in Narrrrcclimated Sewage Sludge after Addition of C h l o ~ t mand Other Toxicants DANIEL H . ZITOMER*tt A N D RICHARD E . SPEECE Environmental and Water Resources Engineering, Vanderbilt University, Nashville, Tennessee 37235
An unacclimated culture from a municipal anaerobic digester transformed chloroform at initial concentrations from 320 to 35OOpglL. Dichloromethane and chloromethane were intermediates, and carbon dioxide was a product. Initial chloroform concentration influenced the transformation rate constant. With 320 p g I L chloroform, gas production was 110 f 4% of theoretical, and the specific firstorder rate constant for chloroform was 4.55 L g-l day.-’ With 80OpglL chloroform, gas production was reduced to 56 f 12% of theoretical, and the first-order rate constant was 1.21 L g-l day.-’ Methanethiol was detected when toxic doses of chloroform were administered; however, no methanethiol was evident at chloroform concentrations of 320 pglL. Also, the addition of 100 mg/L sodium azide and 47 mg/L trichloroethylene resulted in quantifiable methanethiol concentrations. Radiolabeled chloroform (14CHC13) was employed, and the methanethiol detected was not radiolabeled. Therefore, methanethiol was not a product of chloroform, dichloromethane, or chloromethane transformation but was detected in response to a toxic event. These results are in contrast to other reports that methanethiol may be a direct product or intermediate of chloromethane and methyl bromide reductive dehalogenation.
Introduction Although chloroform (CHCl3) is recalcitrant to biological transformation under conventional aerobic conditions, it is transformed under co-metabolic aerobic, anaerobic, and sequential anaerobidaerobic environments ( I ) . Under anaerobic conditions, the reductive dechlorination of CHCL by Acetobacterium woodii has been reported to produce mostly carbon dioxide (Cod,with dichloromethane (CHZClz) and traces of monochloromethane (CH3Cl) identified as intermediates (2). A Methanosarcina sp. was found to reductively dechlorinate CHC13, producing methane in stoichiometric amounts (3). Also, a Clostridium sp. transformed CHC13to CHZC12 and other unknown products ( 4 ) . The biotransformation of less chlorinated homologs is also pertinent since sequential reductive dechlorination is usually the anaerobic degradation mechanism. Recent papers suggest that CH3Cl was transformed to methanethiol (CH3SH)by a mixed anaerobic culture which was enriched to utilize CH2CL as a sole source of carbon and energy (5, 6). The mass of methyl groups recovered in CH3SH,methyl sulfide (CzH&),and CH&l after 3 weeks was between 80 and 90% of the CH3Cl added. The authors conclude that CH3SH and C2H& are direct products of CH3C1degradation. Others have reported that CH3SH and C2H& were transiently detected when biologically active anaerobic soil slurries were exposed to methyl bromide (CH3Br) (7). Interestingly, when the specific biological inhibitors 2-bromoethanesulfonic acid (BES) and molybdate were added to biological soil slurries, the CH3SH concentrations were higher than in uninhibited systems. In inhibited systems, the molar percent of hypothesized intermediates and products recovered, including CH3SH, was greater than 223% of the CH3Br added. We have observed the formation of CH3SH during reductive dechlorination of CHC13 by an unacclimated anaerobic mixed culture. However, radiolabeled carbon from 14CHC13was not detected in the CH3SH produced. In this paper, we present evidence that CH3SHproduction is due to a toxic effect and not from the conversion of CH3C1, CHZCl2,or CHC13.
Experimental Section Chemicals and Radioisotopes. The following chemicals were employed: sodium azide (purified, 99+%; Fisher Scientific Co., Pittsburgh, PA), denatured ethanol (spectrophotometric grade, Aldrich Chemical Co., Milwaukee, WI), dodecane (99%,Aldrich Chemical Co., CHC13 (highpressure liquid chromatography grade, 99.9+%; Aldrich Chemical Co., CH& (certifiedherican Chemical Society grade, 99.9+%; Fisher Scientific Co., Pittsburgh, PA), trichloroethylene (CzHC13) (certified American Chemical Society grade; Fisher Scientific Co.), CH3SH (1-g ampule, 99.0%;Chem Service, West Chester, PA), and CZH~S (Chem Service). Chloromethane was obtained in gaseous form (100-gdisposable cylinder, 99.5+%;Aldrich Chemical Co.). Scintiverse I1 (Fisher Scientific Co.) liquid scintillation cocktail was used. * Author to whom all correspondence should be addressed; Telephone: (615) 252-4400; Fax: (615) 255-6572. + Present address: Barge, Waggoner, Sumner, and Cannon, Inc., 162 3rd Ave. N., Nashville, TN 37201.
762 IENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 3, 1995
0013-936x/95/0929-0762$09.00/0
0 1995 American Chemical Society
Chloroform and CH2C12 standards were prepared in dodecane, while CH3SH and C2H6Sstandards were prepared in denatured ethanol. Methyl sulfoxide (99+%, Aldrich Chemical Co.) was initially employed for CH3SH and C2H& standards. However, trace amounts of the sulfur analytes were present in this solvent, and its use was discontinued. 14CHC13 originated from a commercial source (New England Nuclear, Natick, MA). A 2% vlv stock solution of I4CHC13(1.8 mCilmmo1) in denatured ethanolwas received as a gift and stored at 4 "C in a 1-mL conical vial capped with a Teflon-lined silicon septum. Purity and identity was confirmed by gas chromatography. The solution activity was initially 5.5 x lo8 cpmlmL but decreased to 2.2 x lo8 cpmlmL after 3 months of use. Anaerobic Culture Preparations. The mixed culture employed was obtained from municipal anaerobic digesters of the Murfreesboro,TN, wastewater treatment plant. Serum bottles (160 mL) were chargedwith 50 mL of sludge having avolatile suspended solid (VSS) concentration between 2100 and 10 300 mg/L as determined by standard methods (8). The bottles were flushed with a mixture of N2 and COZ (70:30 vol/vol) after the culture was added and then sealed with Teflon-lined rubber septa and aluminum crimp caps. Abiotic controls were prepared in a similar manner, but 50 mL of deionized water was added instead of culture. All bottles were incubated at 35 "C in the dark on a shaker table. Chloroform concentrations in controls were stable, with no losses apparent during experimental periods. The ICs0 value for a methanogenic culture exposed to CHC13 is 900 pg/L (9). Therefore, the aqueous CHC13 concentration was varied between values higher and lower than 900 pg/L to assess the effects of toxicity on biotransformation, but was always below the solubility limit of approximately 8900 mg/L (10). Three experimental setups may be classified according to the initial aqueous CHC13 concentrations employed. The first setup involved cultures which were not fed a primary substrate and received 4 mL of a 60 mglL aqueous CHC13 solution. This produced an initial aqueous CHC13 concentration of 3500 pglL after equilibration between gas and liquid took place. For the second setup, cultures received 8.0 pL of the 14CHC13stock solution. This produced an initial aqueous 14CHC13concentration of 800 pg/L and an ethanol concentration of 126 mg/L. This is well below the methanogenic ICs0 of 42800 mg/L for ethanol (9). Therefore, ethanol was probably not toxic, but may have been a cosubstrate. In addition, 500 pL of a 100 g/L aqueous glucose solution was provided as a primary substrate. Both active and abiotic systems were run in triplicate. Chloromethanes and CH3SH concentrations were monitored over a 6-day period using gas chromatography. Headspace and aqueous samples were taken on day 0 and day 6 for radioisotope analysis. The third experimental setup was identical to the second, except that the 14CHC13concentration in the stock solution had decraased from 2% vlv to approximately 1% vlv. Therefore, the initial aqueous CHC13 concentration was 320 iug/L. A fourth investigation was run in order to help support the hypothesis that there is a relationship between CH3SH production and toxicity. Sodium azide (NaN3) and trichloroethylene (C2HC13)were added to individual serum bottles containing active cultures which did not receive primary substrate. The NaN3 dose was added as 500 pL of a 10 g/L aqueous NaN3 solution. This produced a 100 mg/L NaN3
aqueous concentration in the serum bottle. The C2HCl3 dose was added as 5 yL of net trichloroethylene which, when corrected for volatility, yielded a 47 mglL aqueous concentration. The methanogenic culture IC50 value for trichloroethylene is 13 mg/L (9). Therefore, toxicity responses were expected to occur from the relatively high dose administered. Analysis of Volatile Organics. A gas chromatograph (GC)with a flame ionization detector (Autosystem,PerkinElmer Corp., Norwalk, CT) was used for analysis. An 8-ft x 0.125-in. 0.d. stainless steel column packed with 1%SP1000 on 60/80 Carbopack-B (Supelco, Inc., Bellefonte, PA) accomplished separation. The helium carrier gas flow rate was 20 mllmin, and the oven temperature was maintained for 5 min at 60 "C, then increased 5 "C/min to 210 "C, and held at 210 "C for 8 min. Retention times of CHC13, C2H& CHzClZ, CH3SH, and CH3C1were 16.7, 9.5, 8.5, 3.0, and 1.7 min, respectively. Cochromatography with authentic material was used to confirm the identity of the compounds, and freshly prepared external gravimetric standards were used for quantification of analytes. The analytical procedure consisted of (1)equilibrating the serum bottle pressure to 1 atm by inserting the needle of a 100-mLglass syringewith a wetted barrel into the bottle septum, (2) taking a 500-yL headspace sample with a microsyringe inserted through the septum, and (3) forcing the excess equilibration gas in the 100-mL syringe back into the serum bottle to prevent loss of the analytes. The 500-pL sample was then directly injected into the GC. The headspace concentrations of analytes were corrected by calculating the true pressure in the serum bottle headspace and converting the 1 atm concentration to the pressurized serum bottle concentration. Dimensionless Henry's constants at 35 "C for CHC13, CHE12, CH3C1, CH3SH, and CZH& of0.23,0.13,0.52,0.18,andO.ll were employed to estimate liquid concentrations. The method detection limits (MDLs) and estimated quantitation limits (EQLs) are presented in Table 1. A detailed description of their determination is presented elsewhere (11). Analysis of Radioisotopes. 14Cactivity was measured using a liquid scintillation counter (LS 3801 Series Liquid Scintillation System,Beckman Instruments, Fullerton, CAI. The counting efficiency was always greater than 95%. Therefore, counts per minute (cpm) were used directly to measure activity. The radioisotope analyses of headspace samples were performed as a qualitative procedure to indicate whether the CH3SH produced was a degradation product of chlorinated methanes or the product of a different biological process. A 500-pL gas sample was injected into the GC while the FID detector was off, and fractions were collected in 20-mL glass scintillation vials at the detector exit. The headspace COz fraction was trapped in 5 mL of deionized water which contained 50 pL of 10 N NaOH (12). Ten milliliters of scintillation cocktail was added before counting. All other fractions were trapped directly in 10 mL of scintillation cocktail. Detector exit gas was collected at 0-1.5-, 1.6-2.7-, 2.8-5.0-, 7.0-9.0-, and 15-18-min fractions to determine the possible distribution of 14Camong COz, CH3C1, CH3SH, CH&, and CHC13 fractions, respectively. A CzHsSfractionwas not collected since FID analysis indicated that it was only present in trace amounts. The recovery of 14C in headspace samples resolved on the GC column was investigated by injecting 2 pL of a solution containing 325 mg/L CHC13 (1.8 mCi/mmol of VOL. 29, NO. 3, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
763
TABLE 1
Gas Chromatography Method Detection Limits (MDLs) and Estimated lluantitation Limits (EQLs) in Various Phasesa MDLs analyte
Henry's constant (unitless at 35 O C )
CH3CI CH3SH CH2CI2 CzHsS CHC13
0.52 0.18 0.13 0.11 0.23
EQLs
headspace
aquaous
total mass
headspace
aquaous
total mass
bdL)
ban)
bmol)
ban)
ban)
(rmol)
1.3 2.7
2.6 15 6.2 31 4.8
5.4 x 10-3 2.2 x 10-2 4.7 10-3 3.1 x 3.0 10-3
4.3 8.6 2.6 11 3.5
8.3 48 20 99 15
7.4 x 2.8 10-2 1.5 x I O - * 9.9 x 10-2 9.5 10-3
0.80 3.4 1.1
a A 500-pL headspace sample was taken from 160-mL serum bottles containing 50 mL of deionized distilled water. Headspace MDLs and EQLs were experimentally determined. Aqueous MDLs and EQLs are estimated using Henry's constant and headspace concentration values. Total mass MDLs and EQLs are estimated from headspace values and Henry's constants.
CHC13) in denatured ethanol onto the GC column and capturing the fraction between 15 and 18 min. Results were compared to the those obtained when 2.0 pL was directly injected into scintillation cocktail. Three experiments yielded an average recovery of 59 & 2% (mean =k SD). Although the recovery was not complete, the precent recovered was reproducible. Quantification of the analytes using radioisotope techniques was not necessary since GCFID analysis was also employed. Nevertheless,the recovery was sufficient to track the possible degradation products. It is probable that CHC13 transfer from the gas phase to the scintillation cocktail was less than loo%, and this limited the recovery. The recovery of CH3SH in the scintillation cocktail was also investigated. Direct GC injection of scintillation cocktail was not feasible due to peak interferences from cocktail constituents. Therefore, a headspace analysis technique was employed. A total of 1500pLof a gas sample containing 250 pg/L CH3SH was injected into 7 mL of scintillation cocktail contained in a 9-mL glass vial with a Teflon-coated butyl rubber septum. No CH3SH was detected when a GC-FID analysis of the vial headspace was immediately performed. If 2% of the CH3SH added had escaped to the headspace, it would have been detectable in the gas sample. Therefore, most of the CH3SH was captured within the cocktail. Radioisotope analysis of aqueous samples was also performed in an effort to quantify the distribution of COz, volatile, and nonvolatile metabolic fractions. Liquid aliquots were filtered through a 0.45-pm pore-size glass fiber filter using a glass syringe and a stainless steel filter holder. Aliquots (0.13mL) were added to 10 mL of cocktail for total counting. Acid and base fraction counting was accomplished by injecting 0.13-mL filtered aliquots into 5 mL of deionized distilled water to which either 30 p L of 6 N HCl or 50pL of 10 N NaOH was added. Acid and base fractions were then purged with NP (120-270 mL/min) for at least 5 min to remove volatile analytes. Then, 10 mL of cocktail was added. Base minus acid fractions were recorded as 14c02(12, 13).
Chemiluminescenceinterferencewith liquid scintillation counting associated with NaOH has been reported previously (14, 1 3 , but chemiluminescence effects were not evident during our investigations. However, to preclude chemiluminescence problems, a verification experiment was run. A 500-pL headspace sample from a sterile control was injected into 20 mL of scintillation cocktail containing 2 mL of deionized water. The results were compared with those obtained when 20 UL of 10 N NaOH was also added 764
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 3,1995
1w-
CH,
90-
80-
I
CHCI,
70-
60u)
5
g i
340. 30-
P10
:0
Y)
D
Y
R
TIME IMINUTES)
FIGURE 1. Day 0 headspace sample GC-FID chromatogram from 160-mL serum bottle containing 50 mL of anaerobic mixed culture (VSS = lo300 m g L initial CHC13 aqueous concentration = 3500 PgR).
to deionized water and scintillation cocktail which received the same 500-pLheadspace aliquot. The mean radioactivity for measurements with NaOH (7749 cpm) and without NaOH (7815 cpm) were not significantly different when measured by the t-test (Y = 10, P = 0.01). Therefore, chemiluminescence interference was negligible.
Results and Discussion Chloroform Transformation. The unacclimated mixture culture transformed CHCl3. Dichloromethane and CH3Cl were detected as intermediates. Figures 1 and 2 present typical chromatographic results from the first experimental setup on day 1 and day 6, respectively. A progress curve representing CHC&transformation results from the second experimental setup is presented in Figure 3. The detection of lesser chlorinated homologs is consistent with previous descriptions of anaerobic reductive dechlorination (3). However, an acclimation time or lag period is commonly observed (16). For example, transformation of chloroform by a pure culture (previously employed to dechlorinate tetrachloroethylene) exhibited a 10-day lag period (3).In contrast, a discernible lag period was not evident during our investigations.
1CQ-
90.
80-
v)
0
R
y1
TIME (MINUTES)
FIGURE 2. Day 6 headspace sample GC-FID chromatogram from 160-mL serum bottle containing 50 mL of anaerobic mixed culture (VSS = lo300 mg/L initial CHC13 aqueous concentration = 35M)
adL1. At an initial concentration of 320 pgIL, CHC13 was reduced to essentially 0 pglL within 3.5 h. Under these conditions, total gas production (methane and carbon dioxide) resulting from the primary substrate was 104 f 4% of theoretical, and no CH3SH was detected. Therefore, no toxic effects were evident. On the other hand, 800pglL of CHC13 caused a toxic effect. Gas production was 56 f 22% of theoretical, and the CHC13 transformation rate constant decreased to 27% of that measured in the uninhibited system. Consideringthese observations,it may be that the ability of unacclimated cultures to reductively dechlorinate has been underestimated because relatively toxic doses of analytes may have been employed. Radioisotope analyses from the second experimental setup demonstrated that carbon dioxide was a product of
CHC13 transformation. The aqueous I4CO2fraction increased from 7 f 4% of the total aqueous radioactivity on day 0 to 24 k 4% on day 6 (Figure 4). It is probable that the radioactivity detected in the COZfraction on day 0 was present as an impurity in the stock solution. Alternatively, it may have been formed during the 30 min between stock solution addition and headspace analysis. The volatile fraction was further divided by converting gas chromatographic data describing CHCl3, CHzC12, and CH3Cl (aqueous concentrations inpmollL) into radioactivity units of cpml mL (Figure 5). The 14CHC13specificity activity of 1.638 x 106 cpmlpmol, measured at the time of the experiment, was used for data conversion. A significant portion of the volatile aqueous radiolabeled fraction was not identified. The unidentified fraction may have been l,2-dichloroethane (CzH4C1z). Others have reported that CzH4C12 was the only significant intermediate present in anaerobic systemswhich biotransformed CHC13 (17). Yet, this product has not generally been observed, and more data are required to validate this mechanism. It is possible that the intermediate, dichloromethane, is transformed into a carbocation (CHZCl+),and two carbocations combine to form C2H4C1z. A nucleophilic unimolecular substitution (&1) in which a carbocation is formed would coincide with a first-order observation of CH3C1transformation (18). In regards to the transformation rate of chlorinated compounds, the initial CHC13 concentration was a factor. Specificfirst-order rate constants were computed by linear regression using chlorinated compound mass vs time data. The CHC13 data employed were from periods when CH2ClZ concentration was below detection and vice versa. The best-fit constant values were divided by the volatile suspended solids (VSS) concentrationto arrive at the specific first-order rate constants. Table 2 presents a synopsis of the analysis. In general, specific first-order constants are influenced by toxicity effects, primary substrates, and temperature. Therefore, the constants presented in Table 2 are not directly comparable since the initial aqueous CHC13 concentration was varied between values near and lower thantheIC50(9OOpglL)and toxicity effects differ. However, the systems receiving CHC13 concentrations of approxi-
Control
_I.....,
.......
I . ”
^ ^
Methanehiol
0.2 0.0
0
1
Y
2
3
Dichloromethane
I Y
”
4 chloromethane
I
1
1
4
5
6
7
8
Time (days) FIGURE 3. Progress curves representing CHCls (800pg/l original aqueous concentration) biotransformation and CHJSH mass increase. Glucose was the primary substrate. The VSS was 8800 m a , and each treatment was run in triplicate. Data from abiotic controls are also presented. Error bars represent 1 SD. Nonvisible error bars are smaller than the plot symbol.
VOL. 29, NO. 3, 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY
765
hl"*c,i*.*.mc.I>,, ,, ,Aci..r
my6.At.&A-s
\I/
.,,,.r
FIGURE 4. Recovery of aqueous radiolabeled metabolic fractions.
Average values from triplicate experiments from active cultures (VSS = 8SW m a ) and abiotic controls are presented
10300
mnn
SZDO
vss
5200
zioo
21m1
"11)
FIGURE 6. CHLH induced by 3%0#gll CHCl, with various culture volatile suspended solid (VSSI concentrations. Duplicate experimental results are presented. Moles of CH,SH after 4 days of incubation is shown as a percentage of the total moles of chloromethanes that were transformed to nondetectableproducts or intermediates.
OaybAhinic Arcngc
O w i l . A c w c \\wage
0:ii ., 1. 1
8 s ~\\ri:iec
FIGURE 5. Recovery of aqueous radiolabeled metabolic fractions with an estimated volatilefraction breakdown. The CH&l and CHICh fractions are estimated from gas chromatographic data which wereconvertedtoaqueous radioactivity units. The specificactivity of the "CHCI, was 1.638 x 106 cpmlpmol:this value was used for dataconversion. Bothactivecultures(VSS=88WmgR)andabiotic controls were employed.and the data presented are averagevalues from triplicate experiments.
mately the ICso value yielded rate constants which were three to four times less than those for systems receiving nontoxic doses. In general, the first-order specific rate constants for CH2C12were 2 orders of magnitude less than constants for CHCI, biotransformation. Methanethlol Concentration Increase. Methanethiol was detected when a relatively toxic dose of CHCl, was administered (Figures 3). However, CH3SH was not detected in any of the samples from the third experimental setup, which employed a relatively nontoxic CHCls dose. The origin of CHsSH was not assumed to be from degradation of CHC13because more CH&H was produced than the total CHCl, degraded (Figure6). Therefore, a mass balance
analysis provided evidence that CH,SH formation was a result of processes other than CHCl, biotransformation. In confirmation of this hypothesis, no radioactivity was found in the CH,SH fraction collected during the second experimental setup (Figure 7). In addition, two other toxicants, sodium azide (NaNd and trichloroethylene (C2HC13),induced CH,SH production, whereas CH3SH was not detected in controls that did not receivea toxicant dose (Figure8). Datawerecollected from triplicateexperimentsonday4. Cultures exposed to NaN,. C2HC13,and CHCll produced 12 f 3.3 pg (mean f SD).3.9 f 0.43 pg, and 29 f 8.6 pg of CH3SH, respectively. Previous reports indicate that CH,SH was produced when an aqueous concentration of 79 mg1L CH,CI was added to enriched mixture cultures (3.The ICso value for a methanogenic mixed culture exposed to CHICl is 50 mg1L (9). Therefore, the previous observations of CHISH production may have been made during toxicity events. The CH3SH and C2HeS concentration increases may not have been the result of CH3CI degradation, but may have been related to toxicity. Similarly, the relatively high concentration of CH3SH observed during CH,Br degradation experiments, which included inhibitory BES and molybdate additions, may have been the result ofa toxic response (7). Apossiblemechanism for therelease ofCH,SH involves regulation of cellular osmotic pressure during toxicityinduced stress. For example, C2H6Srelease during culture
TABLE 2
Chloroform and Dichloromethane: First-Order Rate Constants secondary submaelprimary substrate
ML VSS
IglmLl
initial aq chloroform conc
Ira)
ml gas production
+
(C02 C R )
I% theoreticall
mahanethiol production ips)
specific lint-order rate constam k, IL g-' day-'). base e
Z and In)'
chloroformlglucose 8.8 800 56 (22P 38 118)' 1.21 0.884 19) chloroformlglucose 8.8 300 110 (4P NDa 4.55 0.814 1151 dichloromethanelglucose 8.8 800 56 (22P 38 (18)' 0.015 0.958 1191 dichloromethanelglucose 8.8 300 110 (4P NDb 0.019 0.947 1231 'Value determined from triplicate experiments. Number in parentheses is coefficientof variation ICVI, where CV = lstandard deviationlmeanl x 100. ND = not detected. r' is the coefficient of determination and n is the number of obsewations used to determine the rafe constant.
706 m ENVIRONMENTAL SCIENCE & TECHNOLOGY I VOL. 29. NO. 3. 1995
Time Fraction (minutes) 30-
1 METHANE
26. 26. 24.
5
22.
5
20. 18.
1614 .'
12 i
ul
0
z
0
R
the osmolarity of the growth medium. Under more reduced conditions, CH3SH may be detected and not C&S. We have detected increasing CH3SH concentrations in anaerobic sludge subjected to 11 500 mg/L sodium chloride (data not published). Althoughthe toxic concentrations of CHC13 used in our investigation would nct significantly alter the osmolarity of the sludge, they may have adversely affected osmoregulatory processes. The role of CH3SH and Cp&S in relation to anaerobic bacterial osmoregulation and the effect of sulfide concentration on CH3SH deserves investigation. The fact that the toxic doses of CHCl3, NaN3, and trichloroethylene elicit production of CHSH is of practical importance. For example, odor from anaerobic treatment processes is a major problem, and CH3SH is one of the most odoriferous compounds known (20). Therefore, the production of CH3SH should be controlled. It is evident that CH3SH odor control should include prevention of toxicity events. Also, CH3SHconcentrations in digester gas may be used as an indicator of process performance. For instance, we did not detect CHSH in uninhibited mixed cultures but only detected the compound in systems under toxicityinduced stress. Other compounds have been suggested as indicators of anaerobic digester performance (21). Hydrogen gas level may be an indicator of process upset. However,limits on using this parameter as an early warning indicator have been discussed (22). Similarly, the concentration of carbon monoxide in digester gas has been suggested as a indicator (23). It is possible that CH3SH production or CH3SHproduction in combinationwith other indicators may lead to better early warning indicators and process monitoring strategies.
TIME (MINUTES1
FIGURE 7. Typical chromatograph and average radioactivity measurements in headspace samples. Serum bottles contained initial aqueous 'FHCI3 concentrationsof goOpgR. The active culture VSS was 8800 mgR. The day 0 chromatograph is shown as a dotted line, whereas the day 6 chromatograph is a solid line. No CHCIJ was detected after day 1.
1
"1
Chbrofonn
30
/
a .-
c c
5
5 0
1
2
3
4
5
Time (days)
FIGURE 8. Typical CHsSH mass increase induced by potential toxicants. The active cultures (VSS = 8800 mgR) were exposed to the indicated aqueous concentrations of chloroform (CHC131,sodium azide (NaNd, and trichloroethylene (CzHC13). CHJSHwas detected after the addition of the potential toxicants. An active culture control did not receive a toxicant dose.
of phytoplankton (Hymenomonascurterue)under oxidized conditions has been linked to rapid change in the osmolarity of growth media (19). The rate of C2H6Soutput increased when either inorganic salts or sucrose was used to increase
ConcIusions The unacclimated mixture culture transformed CHC13 at initial concentrations from 320 to 3500 ,ug/L, producing CH2C12,CH3C1, COZ,and unidentified volatile products with no discernible lag period. The initial CHC13 concentration influenced the transformation rate. When relatively nontoxic CHCL initial concentrations were employed, the transformation rate constant was higher than when relatively toxic doses were administered. Methanethiol was not directly produced from degradation of the chlorinated methanes but was detected in response to toxicity. The production of CHSH occurred when the initial CHC13 concentration caused inhibition of gas production but was not formed when nontoxic doses of CHC13were administered. Molar mass balances which include CHC13, CHpClp, CH3C1, and CH3SH indicate that the CH3SH produced was more than the chloromethanes lost, and CHSH was produced when toxic doses of NaN3 and CzHC13 were administered to the mixed culture. In addition, the CH3SH produced in systems which received 14CHC13was not radiolabeled.
Acknowledgments We thank Drs. Kristina Hill and Raymond Burk, Division of Gastroenterology,Vanderbilt UniversityMedical Center, for their gift of radiolabeled chloroform.
Literature Cited (1) Zitomer, D. H.;Speece, R. E. Enuiron. Sci. Technol. 1993, 27, 226-244.
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(2) Egli, C.; Tschan, T.; Scholtz, R.; Cook, A. M.; Leisinger, T. Appl. Environ. Microbiol. 1988, 54, 2819-2824. (3) Mikesell, M. D.; Boyd, S. A. Appl. Environ. Microbiol. 1990, 56, 1198- 1201. (4) Galli, R.; McCarty, P. L.App1. Environ. Microbiol. 1989,55,837844. (5) Braus-Stromeyer, S. A.; Cook, A. M.; Leisinger, T. Environ. Sci. Technol. 1993, 27, 1577-1579. (6) Braus-Stromeyer, S. A.; Hermann, R.; Cook, A. M.; Leisinger, T. Appl. Environ. Microbiol. 1993, 59, 3790-3797. (7) Oremland, R. S.; Miller, L. G.; Strohmaier, F. E. Environ. Sci. Technol. 1994, 28, 514-520. (8) American Public Health Association. Stundurd Methodsfor the Examination of Water and Wastewater, 15th ed.; APHA: Washington, DC, 1985. (9) Blum, D. J. W.; Speece, R. E. J. Water Pollut. Control Fed. 1991, 63, 198-207. (10) Nirmalakhandan, N.; Speece, R. E. Environ. Sci. Technol. 1988, 22, 328-338. (111 Zitomer, D. H. Ph.D. Dissertation, Vanderbilt University, 1994. (12) Fathepure, B. 2.;Vogel, T. M.Appl. Environ. Microbiol. 1991,57, 3418-3422. (13) Freedman, D. L.; Gossett, J. M. Appl. Environ. Microbiol. 1989, 55, 2144-2151. (14) Boero, V. Ph.D. Dissertation, Vanderbilt University, 1992.
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Received for review Iuly 18, 1994. Revised manuscript received October 6, 1994. Accepted November 14, 1994.@ ES940438X @
Abstract published inAdvunceACSAbstructs, December 15,1994.
