APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, p. 450-454 Vol. 57, No. 20099-2240/91/020450-05$02.00/0Copyright ) 1991, American Society for Microbiology
Degradation of Toluene and m-Xylene and Transformation ofo-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, processeffluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, andxylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion oftoluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene wascorrelated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene wereobserved in these initial enrichment cultures. However, m-xylene was degraded by a subculture that wasincubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for thedegradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greaterthan 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of thesecompounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradativecapabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.
Groundwater can become contaminated and undrinkableupon leakage of gasoline from underground storage tanks(13). Contamination is primarily due to the presence ofbenzene, toluene, and xylenes (BTX), these compoundsbeing more soluble than other components of gasoline suchas alkanes and polyaromatic hydrocarbons. Benzene is ofthe most concern because of its association with the devel-opment of leukemia in humans (4).Much research on the biodegradation of BTX has been
initiated in the hope of developing bioremediation technolo-gies to purify BTX-contaminated groundwater. All five BTXcompounds (including the three xylene isomers) have beenfound to be biodegradable under aerobic (1, 6, 14, 23) andanaerobic (7, 9-13, 22, 25) conditions. Aerobic degradationof toluene, p-xylene, and m-xylene has been shown to begenetically encoded by the TOL plasmid (23). Benzene (6,14) and only recently o-xylene (1) have been shown to bedegraded by pure cultures of aerobic bacteria. Anaerobicstudies have been completed in soil columns and micro-cosms under different reducing conditions, including iron-reducing, denitrifying, sulfidogenic, and methanogenic con-ditions. Anaerobic degradation of toluene has beendefinitively shown in pure culture under iron-reducing (12)and denitrifying (24) conditions. The toluene-degrading den-itrifier (24) was also shown to degrade m-xylene. On theother hand, anaerobic degradation of benzene, p-xylene, ando-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 biode-gradable, benzene, p-xylene, and o-xylene have been shownto be biodegradable under mixed culture conditions in cer-tain studies and not in others (5, 8-11, 13, 17, 22). No onefactor, i.e., substrate concentration or composition, temper-ature, 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
* Corresponding author.
have been observed in microcosms, it is uncertain whetherthis activity is sustainable under these conditions. Theorganisms that are responsible for the anaerobic degradationof benzene, p-xylene, and o-xylene may be exceptionallyfastidious in light of these results and the lack of success intheir isolation.
In this study, a variety of sources of inocula were utilizedto enrich for denitrifying bacteria that are potentially capableof anaerobically oxidizing a BTX mixture. These sourcesincluded river sediment, soil, groundwater, anaerobic di-gester sludge, and process effluent. Degradation of individ-ual BTX compounds was analyzed with complete balanceson carbon, nitrogen, and electrons to assess the extent oftheir transformation.
MATERIALS AND METHODS
Sources of inocula. Table 1 lists the seven sources ofinocula 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. Samplesfrom source CAl were centrifuged, and the solids were thensuspended in a small volume of the supernatant so that thesample solids were concentrated by a factor of 5. Samplesfrom the remaining sources (BH and KC) were used asobtained.Growth medium and initiation of enrichment cultures.
Inocula were added in 5-ml aliquots in triplicate to 60-mlserum bottles with 45 ml of a mineral salts medium (amodified version of that described by Taylor et al. ). Aliter of this medium contained the following (unless other-wise noted): 7.9 g of Na2HPO4 7H20, 1.5 g of KH2PO4, 0.3g of NH4Cl, 2.02 g of KNO3 (20 mM), 0.1 g ofMgSO4 7H20, 5 ml of trace elements solution (20), 10 ml ofvitamins solution, and 0.01 g of yeast extract. The traceelements solution contained the following (per liter): 50 gof EDTA, 22 g of ZnSO4 7H20, 5.54 g of CaCl2, 5.06 g ofMnCl2 .4H20, 4.99 g of FeSO4 7H20, 1.1 g of(NH4)6Mo7024 4H20, 1.57 g of CuSO4 5H20, and 1.61 gof CoCl2. This solution was adjusted to a pH of 6.0 with
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DEGRADATION OF BTX BY DENITRIFYING CULTURES
TABLE 1. Sources of inocula used to start the denitrifyingenrichment cultures
Name Type Origin
ER Sediment East River boat marina at 26thStreet in New York City, N.Y.
BH Anaerobic digester Second digester at Berkeleysludge Heights, N.J.
NC Soil Lagoon repository for bottoms froma naphtha cracking process
KC Effluent Activated carbon process forremediation of BTX-contaminatedgroundwater in Michigan
CAl Groundwater, Partially remediated gasoline spillsediments site in California
CA2 Soil Untreated gasoline spill site inCalifornia
CA3 Soil Site of a Stoddard solvent (apetroleum distillate) spill
KOH. The vitamins solution contained the following (perliter): 0.002 g of biotin, 0.002 g of folic acid, 0.01 g ofpyridoxine hydrochloride, 0.005 g of riboflavin, 0.005 g ofthiamine, 0.005 g of nicotinic acid, 0.005 g of pantothenicacid, 0.0001 g of B12, 0.005 g of p-aminobenzoic acid, and0.005 g of thioctic acid. The pH of the medium was adjustedto 7.5 with NaOH. The inoculated media were sparged for 30min with argon that had passed through a column of reducedR3-11 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 metha-nol was added (25 ,ul) to the medium after sparging wascomplete. The bottles were sealed with Teflon-coated butylrubber stoppers (West Co., Lancaster, Pa.) and aluminumcrimps. The resultant methanol concentration was 12 mM,and the resultant concentrations for BTX compounds were100 puM each. Stoichiometric calculations, assuming com-plete mineralization of methanol and BTX, showed that 19mM N03- is required if N03 is reduced to N2. Althoughmethanol was present initially in the enrichment cultures asa carrier for BTX and as a cosubstrate, it was determined tobe unnecessary for toluene and m-xylene degradation andwas not used further. Initial samples were taken, and thecultures were incubated at 30C in a stationary position. Thecultures were supplemented with additional KNO3 or indi-vidual BTX compounds upon depletion. All samples andadditions were via sterile syringes that were flushed withargon. This procedure resulted in puncturing the Tefloncoating on the stoppers. Therefore, the stoppers were re-placed with new ones after sampling to minimize sorptiveBTX losses through the stoppers. Anaerobic conditionswere maintained during stopper replacement by gently flush-ing the headspace with argon.Medium preparation for subcultures. Mineral salts medium
without yeast extract was added to serum bottles, whichwere then sealed with Teflon-coated butyl rubber stoppersand crimps. The bottles were then degassed by evacuatingand then pressurizing the headspace with 67 kPa of argon.The bottles were shaken vigorously to ensure effectivegas-liquid mass transfer of oxygen. The evacuation andfilling procedure was repeated three times. The BTX com-pounds were added after deoxygenation, and then the mediawere inoculated.
Analytical methods. Volumetric gas production data andheadspace composition data were utilized to calculate theproduction of N20 and N2. The volume of gas produced was
measured with a water-lubricated glass syringe that wasflushed with argon. The bottle to be sampled was shaken andthen pierced with the syringe. The gas volume was recordedafter the headspace gas had flowed into the syringe and thepressure had equilibrated. The composition of the gas wasmeasured chromatographically with a gas partitioner (model1200; Fisher Scientific, Pittsburgh, Pa.) equipped with a3.35-m by 4.76-mm column packed with 60/80 mesh 13Xmolecular sieves (Supelco, Bellefonte, Pa.) in series with a1.98-m by 3.18-mm column packed with 80/100 mesh Pora-pak Q (Supelco). The total N2O (which includes gaseous anddissolved N20) was determined from the N20 measured inthe headspace and Henry's constant at 25C (1.71 x 106 mmHg ).CO2 was measured by the addition of 1 ml of cult
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