Vol. 57, No. 2
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, p. 450-454
0099-2240/91/020450-05$02.00/0 Copyright ©) 1991, American Society for Microbiology
Degradation of Toluene and m-Xylene and Transformation of o-Xylene by Denitrifying Enrichment Cultures PATRICK J. EVANS,' DZUNG T. MANG,' AND L. Y. YOUNG12* Departments of Microbiology' and Environmental Medicine,2 New York University Medical Center, New York, New York 10016 Received 12 October 1990/Accepted 4 December 1990
Seven different sources of inocula that included sediments, contaminated soils, groundwater, process effluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, and xylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion of toluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene was correlated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene were observed in these initial enrichment cultures. However, m-xylene was degraded by a subculture that was incubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for the degradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greater than 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of these compounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradative capabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.
have been observed in microcosms, it is uncertain whether this activity is sustainable under these conditions. The organisms that are responsible for the anaerobic degradation of benzene, p-xylene, and o-xylene may be exceptionally fastidious in light of these results and the lack of success in their isolation. In this study, a variety of sources of inocula were utilized to enrich for denitrifying bacteria that are potentially capable of anaerobically oxidizing a BTX mixture. These sources included river sediment, soil, groundwater, anaerobic digester sludge, and process effluent. Degradation of individual BTX compounds was analyzed with complete balances on carbon, nitrogen, and electrons to assess the extent of their transformation.
Groundwater can become contaminated and undrinkable upon leakage of gasoline from underground storage tanks (13). Contamination is primarily due to the presence of benzene, toluene, and xylenes (BTX), these compounds being more soluble than other components of gasoline such as alkanes and polyaromatic hydrocarbons. Benzene is of the most concern because of its association with the development of leukemia in humans (4). Much research on the biodegradation of BTX has been initiated in the hope of developing bioremediation technologies to purify BTX-contaminated groundwater. All five BTX compounds (including the three xylene isomers) have been found to be biodegradable under aerobic (1, 6, 14, 23) and anaerobic (7, 9-13, 22, 25) conditions. Aerobic degradation of toluene, p-xylene, and m-xylene has been shown to be genetically encoded by the TOL plasmid (23). Benzene (6, 14) and only recently o-xylene (1) have been shown to be degraded by pure cultures of aerobic bacteria. Anaerobic studies have been completed in soil columns and microcosms under different reducing conditions, including ironreducing, denitrifying, sulfidogenic, and methanogenic conditions. Anaerobic degradation of toluene has been definitively shown in pure culture under iron-reducing (12) and denitrifying (24) conditions. The toluene-degrading denitrifier (24) was also shown to degrade m-xylene. On the other hand, anaerobic degradation of benzene, p-xylene, and o-xylene has not been observed in pure culture. Although studies of the anaerobic transformation of BTX have consistently shown toluene and m-xylene to be biodegradable, benzene, p-xylene, and o-xylene have been shown to be biodegradable under mixed culture conditions in certain studies and not in others (5, 8-11, 13, 17, 22). No one factor, i.e., substrate concentration or composition, temperature, terminal electron acceptor, or medium composition (i.e., mineral salts or microcosms prepared without media), appears to account for these different results. Interestingly, although losses of benzene under denitrifying conditions *
MATERIALS AND METHODS Sources of inocula. Table 1 lists the seven sources of inocula that were used to start the enrichment cultures. Samples from the soil and sediment sources (ER, NC, CA2, and CA3) were diluted 1:1 or 1:2 (wt/wt) with water. Samples from source CAl were centrifuged, and the solids were then suspended in a small volume of the supernatant so that the sample solids were concentrated by a factor of 5. Samples from the remaining sources (BH and KC) were used as obtained. Growth medium and initiation of enrichment cultures. Inocula were added in 5-ml aliquots in triplicate to 60-ml serum bottles with 45 ml of a mineral salts medium (a modified version of that described by Taylor et al. [19]). A liter of this medium contained the following (unless otherwise noted): 7.9 g of Na2HPO4 7H20, 1.5 g of KH2PO4, 0.3 g of NH4Cl, 2.02 g of KNO3 (20 mM), 0.1 g of MgSO4 7H20, 5 ml of trace elements solution (20), 10 ml of vitamins solution, and 0.01 g of yeast extract. The trace elements solution contained the following (per liter): 50 g of EDTA, 22 g of ZnSO4 7H20, 5.54 g of CaCl2, 5.06 g of MnCl2 .4H20, 4.99 g of FeSO4 7H20, 1.1 g of (NH4)6Mo7024 4H20, 1.57 g of CuSO4 5H20, and 1.61 g of CoCl2. This solution was adjusted to a pH of 6.0 with
Corresponding author. 450
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DEGRADATION OF BTX BY DENITRIFYING CULTURES
TABLE 1. Sources of inocula used to start the denitrifying enrichment cultures Name
Type
ER
Sediment
BH NC
Anaerobic digester sludge Soil
KC
Effluent
CAl Groundwater, sediments CA2 Soil
Origin
East River boat marina at 26th Street in New York City, N.Y. Second digester at Berkeley Heights, N.J. Lagoon repository for bottoms from a naphtha cracking process Activated carbon process for remediation of BTX-contaminated groundwater in Michigan Partially remediated gasoline spill site in California Untreated gasoline spill site in
California CA3
Soil
Site of a Stoddard solvent (a petroleum distillate) spill
KOH. The vitamins solution contained the following (per liter): 0.002 g of biotin, 0.002 g of folic acid, 0.01 g of pyridoxine hydrochloride, 0.005 g of riboflavin, 0.005 g of thiamine, 0.005 g of nicotinic acid, 0.005 g of pantothenic acid, 0.0001 g of B12, 0.005 g of p-aminobenzoic acid, and 0.005 g of thioctic acid. The pH of the medium was adjusted to 7.5 with NaOH. The inoculated media were sparged for 30 min with argon that had passed through a column of reduced R3-11 catalyst (Chemical Dynamics, South Plainfield, N.J.) to remove traces of oxygen. A mixture of benzene, toluene, p-xylene, m-xylene, and o-xylene (200 mM each) in methanol was added (25 ,ul) to the medium after sparging was complete. The bottles were sealed with Teflon-coated butyl rubber stoppers (West Co., Lancaster, Pa.) and aluminum crimps. The resultant methanol concentration was 12 mM, and the resultant concentrations for BTX compounds were 100 puM each. Stoichiometric calculations, assuming complete mineralization of methanol and BTX, showed that 19 mM N03- is required if N03 is reduced to N2. Although methanol was present initially in the enrichment cultures as a carrier for BTX and as a cosubstrate, it was determined to be unnecessary for toluene and m-xylene degradation and was not used further. Initial samples were taken, and the cultures were incubated at 30°C in a stationary position. The cultures were supplemented with additional KNO3 or individual BTX compounds upon depletion. All samples and additions were via sterile syringes that were flushed with argon. This procedure resulted in puncturing the Teflon coating on the stoppers. Therefore, the stoppers were replaced with new ones after sampling to minimize sorptive BTX losses through the stoppers. Anaerobic conditions were maintained during stopper replacement by gently flushing the headspace with argon. Medium preparation for subcultures. Mineral salts medium without yeast extract was added to serum bottles, which were then sealed with Teflon-coated butyl rubber stoppers and crimps. The bottles were then degassed by evacuating and then pressurizing the headspace with 67 kPa of argon. The bottles were shaken vigorously to ensure effective gas-liquid mass transfer of oxygen. The evacuation and filling procedure was repeated three times. The BTX compounds were added after deoxygenation, and then the media were inoculated. Analytical methods. Volumetric gas production data and headspace composition data were utilized to calculate the production of N20 and N2. The volume of gas produced was
451
measured with a water-lubricated glass syringe that was flushed with argon. The bottle to be sampled was shaken and then pierced with the syringe. The gas volume was recorded after the headspace gas had flowed into the syringe and the pressure had equilibrated. The composition of the gas was measured chromatographically with a gas partitioner (model 1200; Fisher Scientific, Pittsburgh, Pa.) equipped with a 3.35-m by 4.76-mm column packed with 60/80 mesh 13X molecular sieves (Supelco, Bellefonte, Pa.) in series with a 1.98-m by 3.18-mm column packed with 80/100 mesh Porapak Q (Supelco). The total N2O (which includes gaseous and dissolved N20) was determined from the N20 measured in the headspace and Henry's constant at 25°C (1.71 x 106 mm Hg [21]). CO2 was measured by the addition of 1 ml of culture medium to a 14-ml sealed tube that contained 100 ,ul of 10 N H2SO4 to convert HCO3- and C032- to CO2. The tube was then shaken to transfer the CO2 to its headspace, which was then assayed for CO2 by gas chromatography. Nitrate and nitrite were measured spectrophotometrically (Hach Co., Loveland, Colo.). The method employs cadmium reduction of nitrate to nitrite, formation of the diazonium salt with sulfanilic acid, and formation of a colored complex with chromotropic acid. The concentrations of the BTX compounds were measured on a gas chromatograph (model 3700; Varian, Sunnyvale, Calif.) with flame ionization detection. A 1.75% Bentone 34-5% SP1200 on Supelcoport (100/120) column (1.83 m by 3.18 mm) was used. The flow rate was 20 ml of nitrogen per min. The temperatures of the injector, detector, and column were 120, 200, and 70°C (isothermal), respectively. Samples (1 ml) were taken anaerobically from the enrichment cultures and extracted in 2-ml vials with 0.4 ml of pentane that contained 1 mM nonane as an internal standard. Aliquots of 1 ,lI of the extracts were injected. Any aromatic hydrocarbon that was adsorbed to sample solids was measured as well as dissolved hydrocarbon, since the samples were not centrifuged or filtered before extraction. Dry cell weight was determined by centrifugation of a culture and washing of the sedimented cells with distilled water. After a second centrifugation, the cells were transferred to a tared aluminum weighing dish with a minimal amount of distilled water and dried at 120°C overnight. The dish that contained the cells was allowed to cool in a desiccator before measurement of the gross weight. RESULTS Fate of BTX in the initial enrichment cultures. All of the enrichment cultures demonstrated losses of 90 to 100% of toluene within 1 to 3 months of incubation, with the exception of one of the ER replicates. A typical example is shown in Fig. 1. Toluene was nearly depleted in the BH cultures by the time the first sample was taken. A second addition of toluene was also effectively depleted by these cultures. Substrate loss was accompanied by loss of nitrate and production of N2. Electron balance determinations were not possible with these initial cultures because of the metabolism of methanol. The decreases in benzene, p-xylene, and m-xylene concentrations (Fig. 1) were largely due to volatilization into the headspace and subsequent loss during stopper replacement. On the other hand, o-xylene depletion, although incomplete, appeared to be greater than that of benzene, p-xylene, and m-xylene. These losses also appeared to occur simultaneously with that of toluene and were repeated upon a
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EVANS ET AL.
452
00 0
ij'
60
loo 0
200
0
20
60
40
80
100
0
120
20
40
Time (d) FIG. 1. Fates of benzene (0), toluene (O), p-xylene (A), in-xylene (V), and o-xylene (-) in the BH enrichment culture. The concentrations are averages for the replicates. Toluene (100 ,uM), methanol (12 mM), and KNO3 (10 mM) were added on day 39. On day 69 the cultures were found to be depleted of N03 and were fed 20 mM KNO3.
second addition of toluene (Fig. 1). Partial depletion of o-xylene concomitant to that of toluene was observed with all inocula. A statistical comparison was made to (i) substantiate a correlation between the o-xylene and toluene losses and (ii) determine that the losses of o-xylene were significantly greater than the losses of benzene, p-xylene, and m-xylene. The loss of compound (i.e., benzene, p-xylene, m-xylene, or o-xylene) between two sample points was calculated for each instance in which the toluene loss was greater than 50 p.M. These data were averaged for each compound and include data from all of the enrichment cultures and replicates (n = 23) (Table 2). Averages were also calculated for the ratio of the loss of benzene or xylene to the loss of toluene for each period of toluene loss greater than 50 ,uM. An analysis of variance (3) of the average losses of benzene, p-xylene, m-xylene, and o-xylene (Table 2) demonstrated that these average losses were not all equal (P < 0.001). Therefore, the loss of o-xylene (Table 2) was greater than any loss of benzene, p-xylene, or m-xylene during a period of toluene depletion. The same conclusion can be drawn for the ratio of compound loss to toluene loss. Metabolism of toluene and o-xylene in a subculture of the CA2 enrichment culture. One of the CA2 culture replicates was diluted into fresh mineral salts medium without yeast extract and twice fed 100 ,uM toluene alone. Toluene was depleted after each addition. This culture was split into 25-ml samples in 27-ml anaerobic tubes with or without toluene
60
80
100
120
Time (h) FIG. 2. Losses of o-xylene in the absence (O) and presence (U) of toluene (0) in a subculture of the CA2 enrichment culture previously grown on toluene. Error bars are shown for data after the initial sample and are + 1 standard deviation. The standard deviation is less than half the width of the symbol in cases that show no error bar.
and supplemented with benzene, p-xylene, m-xylene, or o-xylene, in duplicate for each condition. A sterile control that contained all five BTX compounds was autoclaved after addition of the compounds. The loss of o-xylene was greater in the presence of toluene than that in its absence (Fig. 2). This enhancement of o-xylene depletion appeared to occur during the active metabolism of toluene. In the absence of toluene, negligible depletion of o-xylene occurred relative to that in the sterile control, which contained all five BTX compounds (data not shown). No enhancement of the losses of benzene, p-xylene, or m-xylene was observed in the presence of toluene even though toluene was completely metabolized. The concentrations of these compounds remained the same as those in the sterile control. Toluene metabolism coupled to nitrate reduction. Culture CA2 was further enriched by dilution and subculture on toluene as the sole carbon source. This culture was then used to inoculate (1%, vol/vol) duplicate 150-ml quantities of mineral salts medium without yeast extract, nitrate, or methanol. These cultures were amended as shown in Table 3 and incubated for 7 days. Toluene metabolism was dependent on the presence of nitrate, and denitrification was dependent on the presence of toluene (Table 3). Growth was observed only in the presence of toluene and nitrate. Nitrite can also serve as the sole terminal electron acceptor for toluene oxidation, as evidenced by complete metabolism of toluene and production of nitrous oxide and dinitrogen (data not shown). Carbon, nitrogen, and electron balances for toluene degradation. Table 4 shows balances on carbon, nitrogen, and
TABLE 2. Comparison of losses of benzene and xylenes during periods of substantial toluene loss BTX compound
Toluene Benzene
p-Xylene m-Xylene o-Xylene
Avg loss" (,uM) + SD
86.4 10.9 13.0 12.4 22.7
± ± ± ± ±
13.1 9.1 9.7 9.6 9.2
Loss ratio' ± SD
0.12 0.15 0.14 0.26
± 0.07
+ 0.07 ± 0.05 ± 0.05
" Average losses of benzene or xylene and the concomitant average loss of toluene. Averages are for all inoculum sources. b Average ratios of benzene or xylene loss to the concomitant toluene loss.
TABLE 3. Dependence of toluene loss and associated growth on nitrate of a subculture of enrichment CA2 Initial toluene concn (,uM)
Initial KNO3 concn (mM)
Growth"
300 0 330
0 2 2
-
NAb
+
0
"Determined by visual inspection of turbidity. "NA, Not applicable.
Final toluene concn (tiM)
N2 concn
320
12 12 54
(1iM)
DEGRADATION OF BTX BY DENITRIFYING CULTURES
VOL. 57, 1991
TABLE 4. Balances on carbon, nitrogen, and electrons for a CA2 subculture grown on 300 ,uM toluene Balance
Carbon
Source
Toluene (4.11)
Nitrogen N03- (0.282)
Amt
Sink
NO2 N20
NO3-* N2 Total
e-
2.34 2.46 4.80 0.227 0.0334 0.0845 0.344
CO2 Cells Total NO2N20 N2 Total
Electron Toluene -* CO2 + NO3cells (1.17) NO3-
or
balancea
0.453 0.134 0.423 1.01
% Difference
17.0
22.0
-13.7
a Numbers are milligrams of carbon, millimoles of nitrogen (N), and millimoles of electrons for carbon, nitrogen, and electron balances, respec-
tively.
electrons after the degradation of 300 ,uM toluene as the sole carbon source by the subcultured CA2 enrichment in the presence of 2 mM N03 . CO2 accounted for 57% of the toluene loss. The remainder was accounted for as cell mass on the basis of an assumption that approximately 50% of the cell dry weight is carbon (2). The nitrogen balance includes only nitrogen species involved in electron transport and not those involved in assimilation. The electron balance was based on the following equation to account for assimilation of carbon: C7H8 + 4;8NO3 + 4.8H+ + 0.6NH3 -- 4CO2 + 0.6C5H702N + 2.4N2 + 5.2H20. The stoichiometry of conversion of C7H8 to CO2 in the above equation was established from the carbon balance data in Table 4. The elemental formula for cell mass was taken from McCarty et al. (15). Thus the oxidation of 1 mmol of toluene yielded 24 mmol of electrons. The above equation is based upon no accumulation of N02 or N20; however, the nitrogen balance showed that this basis is incorrect. Thus the millimoles of electrons used for the reduction of NO3- were calculated from the nitrogen balance data in Table 4 and the following stoichiometric factors: 2 mmol of electrons per mmol of N02 produced, 8 mmol of electrons per mmol of N20 produced, and 10 mmol of electrons per mmol of N2 produced. The carbon and nitrogen balances in Table 4 did not completely close, possibly because of inaccuracies inherent to the measurement of the dry cell weight after growth on low substrate concentrations, error in the assumption that 50% of the dry cell weight is carbon, and inaccuracies associated with the assays used for nitrate and nitrite. These errors in turn affected the outcome of the electron balance. m-Xylene degradation. The CA2 subculture was used as an inoculum to generate new cultures with individual BTX compounds as sole sources of carbon rather than a BTX mixture in methanol. The mineral salts medium without yeast extract was used, and the individual BTX compounds (excluding toluene) were added neat at 100 p.M. After 1 week of incubation 50% of the m-xylene was depleted, and after 2 weeks of incubation all of the m-xylene was gone. Nitrous oxide production was also observed in the m-xylene culture. No microbially mediated losses of benzene, p-xylene, or o-xylene or associated production of nitrous oxide were observed. The m-xylene culture was subcultured and grew on m-xylene upon a 10-6 dilution into fresh medium. A subculture was used for a carbon, nitrogen, and electron balance analogous to that for toluene. The balances shown in
453
TABLE 5. Balances on carbon, nitrogen, and electrons for a CA2 subculture grown on 500 ,uM m-xylene Balance
Sink
Source
CO2 Cells Total NO2Nitrogen N03- (1.53) N20 N2 Total Electron m-Xylene -- CO2 N03 - N02 + cells (4.20) NO3- N20 N03 - N2 Total Carbon
m-Xylene (14.4)
Amt
or
balanceae8.14 3.98 12.1 1.47
%
Difference
-16.0
13.7
0.19 0.076 1.74 2.94 0.76 0.38 4.08
-2.9
aSee footnote a of Table 4.
Table 5 are for the degradation of 500 ,uM m-xylene in the presence of 5 mM N03. Carbon dioxide accounted for 57% of the m-xylene consumed. The balance equation that was written to account for assimilation is: C8H10 + 5.6NO3 + 5.6H+ + 0.7NH3 -- 4.5CO2 + 0.7C5H702N + 2.8N2 + 6.4H20. The stoichiometry of conversion of C8H10 to CO2 was established from the data in Table 5, and the electron balance was calculated similarly to that for toluene. The carbon and nitrogen balances possibly did not completely close for the same reasons outlined above for the toluene balance. DISCUSSION The samples obtained for this study encompass a variety of environments obtained from different parts of the United States (Table 1). Most of the samples were from sites known to have been contaminated by specific petroleum products, whereas others (ER, BH) did not have a specific input of contaminant but are considered to have a general input of fuels or industrial effluents. A relatively rapid degradation of toluene was observed with all of these sources (Fig. 1). Furthermore, the toluene-associated partial metabolism of o-xylene was also observed in all of the cultures (Table 2). This suggests that denitrifiers that metabolize toluene and o-xylene may be widespread and can be readily found in the environment. No transformation of benzene, p-xylene, or m-xylene was noted in any of these initial cultures. Although m-xylene degradation was not observed in the original enrichment cultures (Fig. 1), it was observed after subculture of the CA2 enrichment culture on toluene. Thus the m-xylene-degrading bacteria were present in the CA2 inoculum source, but the initial enrichment conditions did not seem to be favorable. m-Xylene was the sole source of carbon when its degradation was observed. Whether the other alkylbenzenes inhibited the m-xylene degraders or whether these microorganisms were unable to compete effectively for nutrients with the toluene degraders is not known at this time. Additionally, the toluene degraders may have m-xylene degradation capabilities that were inactive during the initial incubations. Nevertheless, these results demonstrate the importance of the environmental conditions with respect to the observation of metabolic activities, and may help to explain the varied results on the biodegradability of benzene, p-xylene, and o-xylene found in the literature. The degradation of toluene and m-xylene was quantita-
454
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tively analyzed by means of balances on carbon, nitrogen, and electrons. This is the first demonstration in which all of the substrates and products of oxidation and reduction reactions, including biomass, have been accounted for in the anaerobic biodegradation of aromatic hydrocarbons. Biomass production in denitrifying systems can be considerable and not negligible as in methanogenic or sulfidogenic systems. For example, the cell yield of Paracoccus denitrificans on succinate under denitrifying conditions was 80% of that under aerobic conditions (18). These balances provided evidence that (i) the main products of toluene and m-xylene degradation were CO2 and biomass (Tables 4 and 5); (ii) aromatic ring fission takes place, since more than 50% of the toluene and m-xylene carbon was found as CO2 (Tables 4 and 5); and (iii) the anaerobic oxidation of toluene and m-xylene was stoichiometrically dependent on nitrate reduction and denitrification (Tables 3, 4, and 5). These conclusions are valid and are supported by the data, notwithstanding the fact that some of the balances did not completely close. An accumulation of nitrite was observed during the degradation of toluene and m-xylene. This can be circumvented by decreasing the nitrate concentration to levels that require complete reduction to N2 to account for the complete degradation of toluene or m-xylene (unpublished results). o-Xylene was not metabolized when it was present as the sole source of carbon in denitrifying mixed cultures. The metabolism of o-xylene was dependent on the metabolism of toluene and was biologically mediated (Table 2; Fig. 2). This synergistic effect of toluene was specific to o-xylene, since no analogous losses of benzene or of the other two xylenes were observed. The dependence of o-xylene loss on toluene was not unique to the CA2 enrichment culture, since it was observed in all of the enrichments (Table 2). Transformation of o-xylene in the presence of another degradable substrate has been previously observed (16). In this case Nocardia corallina A-6 transformed o-xylene to o-toluic acid during aerobic growth on hexadecane. This culture also transformed p-xylene to p-toluic acid and 2,3-dihydroxy-4-methylbenzoic acid. The o-xylene transformation that we observed is probably mechanistically different, since no transformation of p-xylene was observed. Studies of pure cultures that degrade toluene and m-xylene are currently underway to facilitate the further characterization of their metabolism and identification of the o-xylene metabolite under these anaerobic conditions. ACKNOWLEDGMENTS
We kindly thank Timothy Vogel for supplying the KC source material, David Kossen and Gene Bolen for the NC and BH source material, and Margaret Findlay at ABB Bioremediation Systems for the CA1, CA2, and CA3 source material. This research was partially supported by NIEHS ES 04895 and the Hazardous Substances Management Research Center of New Jersey. Partial support for P.J.E. was from a National Research Service award (5 T32 AI-07180) from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Baggi, G., P. Barbieri, E. Galli, and S. Tollari. 1987. Isolation of a Pseudomonas stutzeri strain that degrades o-xylene. Appl. Environ. Microbiol. 53:2129-2132. 2. Bailey, J. E., and D. F. Ollis. 1977. Biochemical engineering fundamentals. McGraw-Hill Book Co., New York. 3. Box, G. E. P., W. G. Hunter, and J. S. Hunter. 1978. Statistics for experimenters, an introduction to design, data analysis, and
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Appl. Microbiol. 15:857-865. 17. Reinhard, M., F. Haag, and P. L. McCarty. 1989. Abstr. Proc. Int. Symp. on Processes Governing the Movement and Fate of Contaminants in the Subsurface Environments, Stanford, Calif. 18. Stouthamer, A. H. 1988. Dissimilatory reduction of oxidized nitrogen compounds, p. 245-302. In A. J. B. Zehnder (ed.), Biology of anaerobic microorganisms. John Wiley & Sons, Inc., New York. 19. Taylor, B. F., W. L. Campbell, and I. Chinoy. 1970. Anaerobic degradation of the benzene nucleus by a facultatively anaerobic microorganism. J. Bacteriol. 102:430-437. 20. Vishniac, W., and M. Santer. 1957. The thiobacilli. Bacteriol. Rev. 21:195-213. 21. Washburn, E. W. (ed.). 1928. International critical tables, vol. 3. McGraw-Hill Book Co., New York. 22. Wilson, B. H., G. B. Smith, and J. F. Rees. 1986. Biotransformations of selected alkylbenzenes and halogenated aliphatic hydrocarbons in methanogenic aquifer material: a microcosm study. Environ. Sci. Technol. 20:997-1002. 23. Worsey, M. J., and P. A. Williams. 1975. Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J. Bacteriol. 124:7-13. 24. Zeyer, J., P. Eicher, J. Dolfing, and R. P. Schwarzenbach. 1990. Anaerobic degradation of aromatic hydrocarbons, p.33-40. In D. Kamely, A. Chakrabarty, and G. S. Omenn (ed.), Biotechnology and biodegradation. Gulf Publishing Co., Houston. 25. Zeyer, J., E. P. Kuhn, and R. P. Schwarzenbach. 1986. Rapid microbial mineralization of toluene and 1,3-dimethylbenzene in the absence of molecular oxygen. Appl. Environ. Microbiol. 52:944-947.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, p. 450-454
0099-2240/91/020450-05$02.00/0 Copyright ©) 1991, American Society for Microbiology
Degradation of Toluene and m-Xylene and Transformation of o-Xylene by Denitrifying Enrichment Cultures PATRICK J. EVANS,' DZUNG T. MANG,' AND L. Y. YOUNG12* Departments of Microbiology' and Environmental Medicine,2 New York University Medical Center, New York, New York 10016 Received 12 October 1990/Accepted 4 December 1990
Seven different sources of inocula that included sediments, contaminated soils, groundwater, process effluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, and xylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion of toluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene was correlated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene were observed in these initial enrichment cultures. However, m-xylene was degraded by a subculture that was incubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for the degradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greater than 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of these compounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradative capabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.
have been observed in microcosms, it is uncertain whether this activity is sustainable under these conditions. The organisms that are responsible for the anaerobic degradation of benzene, p-xylene, and o-xylene may be exceptionally fastidious in light of these results and the lack of success in their isolation. In this study, a variety of sources of inocula were utilized to enrich for denitrifying bacteria that are potentially capable of anaerobically oxidizing a BTX mixture. These sources included river sediment, soil, groundwater, anaerobic digester sludge, and process effluent. Degradation of individual BTX compounds was analyzed with complete balances on carbon, nitrogen, and electrons to assess the extent of their transformation.
Groundwater can become contaminated and undrinkable upon leakage of gasoline from underground storage tanks (13). Contamination is primarily due to the presence of benzene, toluene, and xylenes (BTX), these compounds being more soluble than other components of gasoline such as alkanes and polyaromatic hydrocarbons. Benzene is of the most concern because of its association with the development of leukemia in humans (4). Much research on the biodegradation of BTX has been initiated in the hope of developing bioremediation technologies to purify BTX-contaminated groundwater. All five BTX compounds (including the three xylene isomers) have been found to be biodegradable under aerobic (1, 6, 14, 23) and anaerobic (7, 9-13, 22, 25) conditions. Aerobic degradation of toluene, p-xylene, and m-xylene has been shown to be genetically encoded by the TOL plasmid (23). Benzene (6, 14) and only recently o-xylene (1) have been shown to be degraded by pure cultures of aerobic bacteria. Anaerobic studies have been completed in soil columns and microcosms under different reducing conditions, including ironreducing, denitrifying, sulfidogenic, and methanogenic conditions. Anaerobic degradation of toluene has been definitively shown in pure culture under iron-reducing (12) and denitrifying (24) conditions. The toluene-degrading denitrifier (24) was also shown to degrade m-xylene. On the other hand, anaerobic degradation of benzene, p-xylene, and o-xylene has not been observed in pure culture. Although studies of the anaerobic transformation of BTX have consistently shown toluene and m-xylene to be biodegradable, benzene, p-xylene, and o-xylene have been shown to be biodegradable under mixed culture conditions in certain studies and not in others (5, 8-11, 13, 17, 22). No one factor, i.e., substrate concentration or composition, temperature, terminal electron acceptor, or medium composition (i.e., mineral salts or microcosms prepared without media), appears to account for these different results. Interestingly, although losses of benzene under denitrifying conditions *
MATERIALS AND METHODS Sources of inocula. Table 1 lists the seven sources of inocula that were used to start the enrichment cultures. Samples from the soil and sediment sources (ER, NC, CA2, and CA3) were diluted 1:1 or 1:2 (wt/wt) with water. Samples from source CAl were centrifuged, and the solids were then suspended in a small volume of the supernatant so that the sample solids were concentrated by a factor of 5. Samples from the remaining sources (BH and KC) were used as obtained. Growth medium and initiation of enrichment cultures. Inocula were added in 5-ml aliquots in triplicate to 60-ml serum bottles with 45 ml of a mineral salts medium (a modified version of that described by Taylor et al. [19]). A liter of this medium contained the following (unless otherwise noted): 7.9 g of Na2HPO4 7H20, 1.5 g of KH2PO4, 0.3 g of NH4Cl, 2.02 g of KNO3 (20 mM), 0.1 g of MgSO4 7H20, 5 ml of trace elements solution (20), 10 ml of vitamins solution, and 0.01 g of yeast extract. The trace elements solution contained the following (per liter): 50 g of EDTA, 22 g of ZnSO4 7H20, 5.54 g of CaCl2, 5.06 g of MnCl2 .4H20, 4.99 g of FeSO4 7H20, 1.1 g of (NH4)6Mo7024 4H20, 1.57 g of CuSO4 5H20, and 1.61 g of CoCl2. This solution was adjusted to a pH of 6.0 with
Corresponding author. 450
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DEGRADATION OF BTX BY DENITRIFYING CULTURES
TABLE 1. Sources of inocula used to start the denitrifying enrichment cultures Name
Type
ER
Sediment
BH NC
Anaerobic digester sludge Soil
KC
Effluent
CAl Groundwater, sediments CA2 Soil
Origin
East River boat marina at 26th Street in New York City, N.Y. Second digester at Berkeley Heights, N.J. Lagoon repository for bottoms from a naphtha cracking process Activated carbon process for remediation of BTX-contaminated groundwater in Michigan Partially remediated gasoline spill site in California Untreated gasoline spill site in
California CA3
Soil
Site of a Stoddard solvent (a petroleum distillate) spill
KOH. The vitamins solution contained the following (per liter): 0.002 g of biotin, 0.002 g of folic acid, 0.01 g of pyridoxine hydrochloride, 0.005 g of riboflavin, 0.005 g of thiamine, 0.005 g of nicotinic acid, 0.005 g of pantothenic acid, 0.0001 g of B12, 0.005 g of p-aminobenzoic acid, and 0.005 g of thioctic acid. The pH of the medium was adjusted to 7.5 with NaOH. The inoculated media were sparged for 30 min with argon that had passed through a column of reduced R3-11 catalyst (Chemical Dynamics, South Plainfield, N.J.) to remove traces of oxygen. A mixture of benzene, toluene, p-xylene, m-xylene, and o-xylene (200 mM each) in methanol was added (25 ,ul) to the medium after sparging was complete. The bottles were sealed with Teflon-coated butyl rubber stoppers (West Co., Lancaster, Pa.) and aluminum crimps. The resultant methanol concentration was 12 mM, and the resultant concentrations for BTX compounds were 100 puM each. Stoichiometric calculations, assuming complete mineralization of methanol and BTX, showed that 19 mM N03- is required if N03 is reduced to N2. Although methanol was present initially in the enrichment cultures as a carrier for BTX and as a cosubstrate, it was determined to be unnecessary for toluene and m-xylene degradation and was not used further. Initial samples were taken, and the cultures were incubated at 30°C in a stationary position. The cultures were supplemented with additional KNO3 or individual BTX compounds upon depletion. All samples and additions were via sterile syringes that were flushed with argon. This procedure resulted in puncturing the Teflon coating on the stoppers. Therefore, the stoppers were replaced with new ones after sampling to minimize sorptive BTX losses through the stoppers. Anaerobic conditions were maintained during stopper replacement by gently flushing the headspace with argon. Medium preparation for subcultures. Mineral salts medium without yeast extract was added to serum bottles, which were then sealed with Teflon-coated butyl rubber stoppers and crimps. The bottles were then degassed by evacuating and then pressurizing the headspace with 67 kPa of argon. The bottles were shaken vigorously to ensure effective gas-liquid mass transfer of oxygen. The evacuation and filling procedure was repeated three times. The BTX compounds were added after deoxygenation, and then the media were inoculated. Analytical methods. Volumetric gas production data and headspace composition data were utilized to calculate the production of N20 and N2. The volume of gas produced was
451
measured with a water-lubricated glass syringe that was flushed with argon. The bottle to be sampled was shaken and then pierced with the syringe. The gas volume was recorded after the headspace gas had flowed into the syringe and the pressure had equilibrated. The composition of the gas was measured chromatographically with a gas partitioner (model 1200; Fisher Scientific, Pittsburgh, Pa.) equipped with a 3.35-m by 4.76-mm column packed with 60/80 mesh 13X molecular sieves (Supelco, Bellefonte, Pa.) in series with a 1.98-m by 3.18-mm column packed with 80/100 mesh Porapak Q (Supelco). The total N2O (which includes gaseous and dissolved N20) was determined from the N20 measured in the headspace and Henry's constant at 25°C (1.71 x 106 mm Hg [21]). CO2 was measured by the addition of 1 ml of culture medium to a 14-ml sealed tube that contained 100 ,ul of 10 N H2SO4 to convert HCO3- and C032- to CO2. The tube was then shaken to transfer the CO2 to its headspace, which was then assayed for CO2 by gas chromatography. Nitrate and nitrite were measured spectrophotometrically (Hach Co., Loveland, Colo.). The method employs cadmium reduction of nitrate to nitrite, formation of the diazonium salt with sulfanilic acid, and formation of a colored complex with chromotropic acid. The concentrations of the BTX compounds were measured on a gas chromatograph (model 3700; Varian, Sunnyvale, Calif.) with flame ionization detection. A 1.75% Bentone 34-5% SP1200 on Supelcoport (100/120) column (1.83 m by 3.18 mm) was used. The flow rate was 20 ml of nitrogen per min. The temperatures of the injector, detector, and column were 120, 200, and 70°C (isothermal), respectively. Samples (1 ml) were taken anaerobically from the enrichment cultures and extracted in 2-ml vials with 0.4 ml of pentane that contained 1 mM nonane as an internal standard. Aliquots of 1 ,lI of the extracts were injected. Any aromatic hydrocarbon that was adsorbed to sample solids was measured as well as dissolved hydrocarbon, since the samples were not centrifuged or filtered before extraction. Dry cell weight was determined by centrifugation of a culture and washing of the sedimented cells with distilled water. After a second centrifugation, the cells were transferred to a tared aluminum weighing dish with a minimal amount of distilled water and dried at 120°C overnight. The dish that contained the cells was allowed to cool in a desiccator before measurement of the gross weight. RESULTS Fate of BTX in the initial enrichment cultures. All of the enrichment cultures demonstrated losses of 90 to 100% of toluene within 1 to 3 months of incubation, with the exception of one of the ER replicates. A typical example is shown in Fig. 1. Toluene was nearly depleted in the BH cultures by the time the first sample was taken. A second addition of toluene was also effectively depleted by these cultures. Substrate loss was accompanied by loss of nitrate and production of N2. Electron balance determinations were not possible with these initial cultures because of the metabolism of methanol. The decreases in benzene, p-xylene, and m-xylene concentrations (Fig. 1) were largely due to volatilization into the headspace and subsequent loss during stopper replacement. On the other hand, o-xylene depletion, although incomplete, appeared to be greater than that of benzene, p-xylene, and m-xylene. These losses also appeared to occur simultaneously with that of toluene and were repeated upon a
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452
00 0
ij'
60
loo 0
200
0
20
60
40
80
100
0
120
20
40
Time (d) FIG. 1. Fates of benzene (0), toluene (O), p-xylene (A), in-xylene (V), and o-xylene (-) in the BH enrichment culture. The concentrations are averages for the replicates. Toluene (100 ,uM), methanol (12 mM), and KNO3 (10 mM) were added on day 39. On day 69 the cultures were found to be depleted of N03 and were fed 20 mM KNO3.
second addition of toluene (Fig. 1). Partial depletion of o-xylene concomitant to that of toluene was observed with all inocula. A statistical comparison was made to (i) substantiate a correlation between the o-xylene and toluene losses and (ii) determine that the losses of o-xylene were significantly greater than the losses of benzene, p-xylene, and m-xylene. The loss of compound (i.e., benzene, p-xylene, m-xylene, or o-xylene) between two sample points was calculated for each instance in which the toluene loss was greater than 50 p.M. These data were averaged for each compound and include data from all of the enrichment cultures and replicates (n = 23) (Table 2). Averages were also calculated for the ratio of the loss of benzene or xylene to the loss of toluene for each period of toluene loss greater than 50 ,uM. An analysis of variance (3) of the average losses of benzene, p-xylene, m-xylene, and o-xylene (Table 2) demonstrated that these average losses were not all equal (P < 0.001). Therefore, the loss of o-xylene (Table 2) was greater than any loss of benzene, p-xylene, or m-xylene during a period of toluene depletion. The same conclusion can be drawn for the ratio of compound loss to toluene loss. Metabolism of toluene and o-xylene in a subculture of the CA2 enrichment culture. One of the CA2 culture replicates was diluted into fresh mineral salts medium without yeast extract and twice fed 100 ,uM toluene alone. Toluene was depleted after each addition. This culture was split into 25-ml samples in 27-ml anaerobic tubes with or without toluene
60
80
100
120
Time (h) FIG. 2. Losses of o-xylene in the absence (O) and presence (U) of toluene (0) in a subculture of the CA2 enrichment culture previously grown on toluene. Error bars are shown for data after the initial sample and are + 1 standard deviation. The standard deviation is less than half the width of the symbol in cases that show no error bar.
and supplemented with benzene, p-xylene, m-xylene, or o-xylene, in duplicate for each condition. A sterile control that contained all five BTX compounds was autoclaved after addition of the compounds. The loss of o-xylene was greater in the presence of toluene than that in its absence (Fig. 2). This enhancement of o-xylene depletion appeared to occur during the active metabolism of toluene. In the absence of toluene, negligible depletion of o-xylene occurred relative to that in the sterile control, which contained all five BTX compounds (data not shown). No enhancement of the losses of benzene, p-xylene, or m-xylene was observed in the presence of toluene even though toluene was completely metabolized. The concentrations of these compounds remained the same as those in the sterile control. Toluene metabolism coupled to nitrate reduction. Culture CA2 was further enriched by dilution and subculture on toluene as the sole carbon source. This culture was then used to inoculate (1%, vol/vol) duplicate 150-ml quantities of mineral salts medium without yeast extract, nitrate, or methanol. These cultures were amended as shown in Table 3 and incubated for 7 days. Toluene metabolism was dependent on the presence of nitrate, and denitrification was dependent on the presence of toluene (Table 3). Growth was observed only in the presence of toluene and nitrate. Nitrite can also serve as the sole terminal electron acceptor for toluene oxidation, as evidenced by complete metabolism of toluene and production of nitrous oxide and dinitrogen (data not shown). Carbon, nitrogen, and electron balances for toluene degradation. Table 4 shows balances on carbon, nitrogen, and
TABLE 2. Comparison of losses of benzene and xylenes during periods of substantial toluene loss BTX compound
Toluene Benzene
p-Xylene m-Xylene o-Xylene
Avg loss" (,uM) + SD
86.4 10.9 13.0 12.4 22.7
± ± ± ± ±
13.1 9.1 9.7 9.6 9.2
Loss ratio' ± SD
0.12 0.15 0.14 0.26
± 0.07
+ 0.07 ± 0.05 ± 0.05
" Average losses of benzene or xylene and the concomitant average loss of toluene. Averages are for all inoculum sources. b Average ratios of benzene or xylene loss to the concomitant toluene loss.
TABLE 3. Dependence of toluene loss and associated growth on nitrate of a subculture of enrichment CA2 Initial toluene concn (,uM)
Initial KNO3 concn (mM)
Growth"
300 0 330
0 2 2
-
NAb
+
0
"Determined by visual inspection of turbidity. "NA, Not applicable.
Final toluene concn (tiM)
N2 concn
320
12 12 54
(1iM)
DEGRADATION OF BTX BY DENITRIFYING CULTURES
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TABLE 4. Balances on carbon, nitrogen, and electrons for a CA2 subculture grown on 300 ,uM toluene Balance
Carbon
Source
Toluene (4.11)
Nitrogen N03- (0.282)
Amt
Sink
NO2 N20
NO3-* N2 Total
e-
2.34 2.46 4.80 0.227 0.0334 0.0845 0.344
CO2 Cells Total NO2N20 N2 Total
Electron Toluene -* CO2 + NO3cells (1.17) NO3-
or
balancea
0.453 0.134 0.423 1.01
% Difference
17.0
22.0
-13.7
a Numbers are milligrams of carbon, millimoles of nitrogen (N), and millimoles of electrons for carbon, nitrogen, and electron balances, respec-
tively.
electrons after the degradation of 300 ,uM toluene as the sole carbon source by the subcultured CA2 enrichment in the presence of 2 mM N03 . CO2 accounted for 57% of the toluene loss. The remainder was accounted for as cell mass on the basis of an assumption that approximately 50% of the cell dry weight is carbon (2). The nitrogen balance includes only nitrogen species involved in electron transport and not those involved in assimilation. The electron balance was based on the following equation to account for assimilation of carbon: C7H8 + 4;8NO3 + 4.8H+ + 0.6NH3 -- 4CO2 + 0.6C5H702N + 2.4N2 + 5.2H20. The stoichiometry of conversion of C7H8 to CO2 in the above equation was established from the carbon balance data in Table 4. The elemental formula for cell mass was taken from McCarty et al. (15). Thus the oxidation of 1 mmol of toluene yielded 24 mmol of electrons. The above equation is based upon no accumulation of N02 or N20; however, the nitrogen balance showed that this basis is incorrect. Thus the millimoles of electrons used for the reduction of NO3- were calculated from the nitrogen balance data in Table 4 and the following stoichiometric factors: 2 mmol of electrons per mmol of N02 produced, 8 mmol of electrons per mmol of N20 produced, and 10 mmol of electrons per mmol of N2 produced. The carbon and nitrogen balances in Table 4 did not completely close, possibly because of inaccuracies inherent to the measurement of the dry cell weight after growth on low substrate concentrations, error in the assumption that 50% of the dry cell weight is carbon, and inaccuracies associated with the assays used for nitrate and nitrite. These errors in turn affected the outcome of the electron balance. m-Xylene degradation. The CA2 subculture was used as an inoculum to generate new cultures with individual BTX compounds as sole sources of carbon rather than a BTX mixture in methanol. The mineral salts medium without yeast extract was used, and the individual BTX compounds (excluding toluene) were added neat at 100 p.M. After 1 week of incubation 50% of the m-xylene was depleted, and after 2 weeks of incubation all of the m-xylene was gone. Nitrous oxide production was also observed in the m-xylene culture. No microbially mediated losses of benzene, p-xylene, or o-xylene or associated production of nitrous oxide were observed. The m-xylene culture was subcultured and grew on m-xylene upon a 10-6 dilution into fresh medium. A subculture was used for a carbon, nitrogen, and electron balance analogous to that for toluene. The balances shown in
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TABLE 5. Balances on carbon, nitrogen, and electrons for a CA2 subculture grown on 500 ,uM m-xylene Balance
Sink
Source
CO2 Cells Total NO2Nitrogen N03- (1.53) N20 N2 Total Electron m-Xylene -- CO2 N03 - N02 + cells (4.20) NO3- N20 N03 - N2 Total Carbon
m-Xylene (14.4)
Amt
or
balanceae8.14 3.98 12.1 1.47
%
Difference
-16.0
13.7
0.19 0.076 1.74 2.94 0.76 0.38 4.08
-2.9
aSee footnote a of Table 4.
Table 5 are for the degradation of 500 ,uM m-xylene in the presence of 5 mM N03. Carbon dioxide accounted for 57% of the m-xylene consumed. The balance equation that was written to account for assimilation is: C8H10 + 5.6NO3 + 5.6H+ + 0.7NH3 -- 4.5CO2 + 0.7C5H702N + 2.8N2 + 6.4H20. The stoichiometry of conversion of C8H10 to CO2 was established from the data in Table 5, and the electron balance was calculated similarly to that for toluene. The carbon and nitrogen balances possibly did not completely close for the same reasons outlined above for the toluene balance. DISCUSSION The samples obtained for this study encompass a variety of environments obtained from different parts of the United States (Table 1). Most of the samples were from sites known to have been contaminated by specific petroleum products, whereas others (ER, BH) did not have a specific input of contaminant but are considered to have a general input of fuels or industrial effluents. A relatively rapid degradation of toluene was observed with all of these sources (Fig. 1). Furthermore, the toluene-associated partial metabolism of o-xylene was also observed in all of the cultures (Table 2). This suggests that denitrifiers that metabolize toluene and o-xylene may be widespread and can be readily found in the environment. No transformation of benzene, p-xylene, or m-xylene was noted in any of these initial cultures. Although m-xylene degradation was not observed in the original enrichment cultures (Fig. 1), it was observed after subculture of the CA2 enrichment culture on toluene. Thus the m-xylene-degrading bacteria were present in the CA2 inoculum source, but the initial enrichment conditions did not seem to be favorable. m-Xylene was the sole source of carbon when its degradation was observed. Whether the other alkylbenzenes inhibited the m-xylene degraders or whether these microorganisms were unable to compete effectively for nutrients with the toluene degraders is not known at this time. Additionally, the toluene degraders may have m-xylene degradation capabilities that were inactive during the initial incubations. Nevertheless, these results demonstrate the importance of the environmental conditions with respect to the observation of metabolic activities, and may help to explain the varied results on the biodegradability of benzene, p-xylene, and o-xylene found in the literature. The degradation of toluene and m-xylene was quantita-
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tively analyzed by means of balances on carbon, nitrogen, and electrons. This is the first demonstration in which all of the substrates and products of oxidation and reduction reactions, including biomass, have been accounted for in the anaerobic biodegradation of aromatic hydrocarbons. Biomass production in denitrifying systems can be considerable and not negligible as in methanogenic or sulfidogenic systems. For example, the cell yield of Paracoccus denitrificans on succinate under denitrifying conditions was 80% of that under aerobic conditions (18). These balances provided evidence that (i) the main products of toluene and m-xylene degradation were CO2 and biomass (Tables 4 and 5); (ii) aromatic ring fission takes place, since more than 50% of the toluene and m-xylene carbon was found as CO2 (Tables 4 and 5); and (iii) the anaerobic oxidation of toluene and m-xylene was stoichiometrically dependent on nitrate reduction and denitrification (Tables 3, 4, and 5). These conclusions are valid and are supported by the data, notwithstanding the fact that some of the balances did not completely close. An accumulation of nitrite was observed during the degradation of toluene and m-xylene. This can be circumvented by decreasing the nitrate concentration to levels that require complete reduction to N2 to account for the complete degradation of toluene or m-xylene (unpublished results). o-Xylene was not metabolized when it was present as the sole source of carbon in denitrifying mixed cultures. The metabolism of o-xylene was dependent on the metabolism of toluene and was biologically mediated (Table 2; Fig. 2). This synergistic effect of toluene was specific to o-xylene, since no analogous losses of benzene or of the other two xylenes were observed. The dependence of o-xylene loss on toluene was not unique to the CA2 enrichment culture, since it was observed in all of the enrichments (Table 2). Transformation of o-xylene in the presence of another degradable substrate has been previously observed (16). In this case Nocardia corallina A-6 transformed o-xylene to o-toluic acid during aerobic growth on hexadecane. This culture also transformed p-xylene to p-toluic acid and 2,3-dihydroxy-4-methylbenzoic acid. The o-xylene transformation that we observed is probably mechanistically different, since no transformation of p-xylene was observed. Studies of pure cultures that degrade toluene and m-xylene are currently underway to facilitate the further characterization of their metabolism and identification of the o-xylene metabolite under these anaerobic conditions. ACKNOWLEDGMENTS
We kindly thank Timothy Vogel for supplying the KC source material, David Kossen and Gene Bolen for the NC and BH source material, and Margaret Findlay at ABB Bioremediation Systems for the CA1, CA2, and CA3 source material. This research was partially supported by NIEHS ES 04895 and the Hazardous Substances Management Research Center of New Jersey. Partial support for P.J.E. was from a National Research Service award (5 T32 AI-07180) from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Baggi, G., P. Barbieri, E. Galli, and S. Tollari. 1987. Isolation of a Pseudomonas stutzeri strain that degrades o-xylene. Appl. Environ. Microbiol. 53:2129-2132. 2. Bailey, J. E., and D. F. Ollis. 1977. Biochemical engineering fundamentals. McGraw-Hill Book Co., New York. 3. Box, G. E. P., W. G. Hunter, and J. S. Hunter. 1978. Statistics for experimenters, an introduction to design, data analysis, and
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