BIODEGRADATION OF BENZENE, TOLUENE, ETHYLBENZENE AND XYLENES IN GAS-CONDENSATE-CONTAMINATED
GROUND-WATER
Philip Morgan Shell Internationale Petroleum Mij. B. V., PO Box 162, 2501 AN The Hague, The Netherlands
Stephen T. Lewis & Robert J. Watkinson* Shell Research Ltd, Sittingbourne Research Centre, Sittingbourne, Kent, UK, ME9 8AG
(Received 8 January 1992; accepted 5 August 1992)
Abstract
The rate and extent of biodegradation o f benzene, toluene, ethylbenzene and xylenes (BTEX) in ground- water was studied in samples from a contaminated site which contained total B T E X concentrations of up to 20 000 I~g litre 1. All compounds were rapidly degraded under natural aerobic conditions. Elevation of incubation temperature, supply o f organic nutrients or addition of inorganic fertiliser did not increase the rate or extent of biodegradation and it appeared that oxygen supply was the factor limiting B T E X degradation at this site. Attempts to increase the dissolved oxygen concentration in the ground-water by the addition of hydrogen peroxide to give a final concentration of 200 mg litre l resulted in the complete inhibition o f biodegradation. No biodegra- dation occurred under anaerobic conditions except when nitrate was provided as a terminal electron acceptor for microbial respiration. Under denitrifying conditions there was apparent biodegradation o f benzene, toluene, e thyl benzene, m-xylene and p-xylene but o-xylene was not degraded. Degradation under denitrifying conditions occurred at a much slower rate than under oxygenated conditions.
INTRODUCTION
Bioremediation (also termed biorestoration or in situ biotreatment) is a promising technology for the clean- up of contaminated soil and ground-water. The princi- ples are simple--conditions in the environment are optimised so that biodegradation of the contaminants can occur at the maximum rate possible (Morgan & Watkinson, 1989). Being so widely used, benzene, toluene, ethylbenzene and xylenes (BTEX) represent key target compounds for bioremediation. Therefore, understanding their biodegradability is important in as-
sessing both their potential for bioremediation and their environmental fate.
In general, it has been found that BTEX components are biodegraded in soil and ground-water under aerobic conditions and may often be biodegraded under anaerobic denitrifying conditions. In some cases anaerobic biodegradation of at least some compounds in the absence of nitrate has been observed. However, the observed responses vary from site to site. Previous reports are summarised in Table 1 and it is apparent from this that a diverse range of results has been obtained. In those experiments employing materials collected or studied in the field, the differences between results indicate that the responses of a microbial popu- lation are site-specific. This may be due to any number of environmental factors. In this paper a study is reported of the effects of environmental conditions on the biodegradation of BTEX in ground-water resulting from a spillage of condensate at a gas pro-duction site.
MATERIALS AND METHODS
Sampling location Ground-water samples were obtained from a natural gas production site at Uiterburen, The Netherlands. A spillage of gas condensate at the site had resulted in a plume of BTEX in the ground-water (Fig 1). The aquifer consisted of medium to coarse sand and the ground-water table was at approximately 3 m depth. Effective containment of the ground-water had pre- vented contamination moving beyond the site bound- aries and remediation trials were ongoing in selected portions of the site. Further details of the location, the contamination and the remediation trials are given by Boks et al. (1990).
Ground-water sampling Ground-water samples were taken from three pre-
existing wells (Fig. 1). Before sampling each well was emptied by pumping and allowed to refill from the
182 P. M o r g a n , S. T. L e w i s , R. J. W a t k i n s o n
Table 1. Summary of previous key papers related to BTEX biodegradation in soil and ground-water
Reference Study Conditions Compounds ~ Findings/comments
Karlson & Lab. Fankenberger (1989) Armstrong et al. (1991) Lab.
Wilson et aL (1983) Lab.
Ridgway et al. (1990) Lab.
Harrison & Barker (1987) Lab.
Berwanger & Barker (1988) Lab.
Chiang et al. (1987), (1989) Lab. Field
Thomas et al. (1990) Lab.
Berg et al. (1990) Lab.
Barker et al. (1987) Field Kuhn et al. (1985, 1988) Lab. Zeyer et al. (1986) Lab. Berry-Spark & Barker (1987) Lab.
Major et al. (1988) Lab.
Hutchins (1991a, b); Lab. Hutchins et al. (1991a-c) Field
Gersberg et al. (1991) Lab.
Battersby & Lab. Wilson (1989) Wilson et al. (1986) Lab.
Gasoline-contam. aquifer Methanogenic B Not biodegraded
Anaerobic sludge Methanogenic BTEoX Biodegraded
Very slow, long lag Methanogenic BT Biodegraded
Very slow, long lag Metabolic pathway determined Anaerobic sludge
Methanogenic ToX Biodegraded BEmXpX Not biodegraded
Gasoline-contam. aquifer Iron-reducing T Biodegraded
BEX Not biodegraded Sulphate-reducing T Biodegraded
BEX Not biodegraded Iron-reducing T Biodegraded
Pure culture Sulphate-reducing T Biodegraded
BEX Not biodegraded
a B, benzene; T, toluene; E, ethylbenzene; oX, ortho-xylene; reX, meta-xylene; p X , para-xylene; X, all xylene isomers (used when there is no difference between isomers or when isomers were not studied individually).
aquifer. Sterile poly(tetrafluoroethane) (PTFE) tubing was introduced and samples taken by means of a peri- staltic pump into sterile 1-1itre glass bottles. Care was taken to ensure that the liquid flow was smooth so that bubbles did not form in the water. The bottles were filled to the brim with water and sealed with PTFE- lined caps in such a way that no air was trapped in them. The samples were returned to the laboratory at 4°C and used as quickly as possible.
Experimental design, set-up and incubation All experiments were performed in glass serum vials sealed with PTFE-lined butyl rubber stoppers. When totally filled to ensure no air entrapment, the volume of water in each vial was 22.5 ml. Biodegradation under natural aerobic, oxygen-supplemented (with hydrogen peroxide), anaerobic and anaerobic denitrify- ing conditions was studied in a total of 20 different treatments. The details of the treatments are sum-
Biodegradation in gas-condensate-contaminated ground water 183
Well 1 /
Gro~ w e l e r flow~
~ ~ Tank park area
i ~\~Well 3
Original plume of contamination {primarily benzene)
I I Approx. 30 m Fig. I. Map illustrating the approximate positions of the
BTEX plume and the ground-water sampling wells used.
marised in Table 2. When it was necessary for supple- ments to be added to the treatments, they were dis- pensed from concentrated stock solutions into pre-cleaned vials and water removed by incubating at 105°C for 18 h. This technique could not be employed when the additive was hydrogen peroxide or anaerobic redox poiser/indicator solution (composition given in Table 2). In these treatments, a minimal volume of stock solution was added to the vials immediately be- fore they were filled with the water samples. Vials to be incubated under anaerobic conditions were filled in an anaerobic cabinet. After filling, vials were shaken to
ensure that the additives had dissolved and were incubated in darkness at 15 or 25°C. At the time of experimental set-up and at intervals thereafter, replicate samples of each treatment were taken for analysis. Vials were de-capped, supplemented with 100 /zl 2% mercuric chloride (HgC12) solution and immediately re-capped. The vials were stored at 4°C and analysed as soon as possible. Concurrent killed control samples were prepared for selected treatments in the same way as described above except that these vials were supple- mented with HgC12 to give a final concentration of approximately 0.02% (w/v).
Analysis of B T E X Contaminant concentration was determined by a purge and trap procedure based on Method 602 of US EPA (1984). Samples (5 ml) of water were added to purge and trap vessels. These were sparged at 20°C with high-purity nitrogen gas for 11 min at a flow rate of 40 + 1 ml rain -1 and volatiles in the off-gas were trapped by passing the effluent gas through glass tubes con- taining 0.2 g Tenax TA resin (Chrompack). BTEX analysis was performed by means of a Carlo Erba thermal desorption autosampler linked to a Vega GC600 gas chromatograph. The trapping tubes were placed into the auto-sampler so that the compounds were back-flushed from the resin. For desorption the follow- ing programme was employed with a helium gas flow of 8 ml min-~:
dry purge (water removal) 2 min at 20°C desorb 5 min at 180°C tube clean 30 min at 180°C
Table 2. Experimental treatments performed to study the degradation of BTEX in ground-water samples under different environmental conditions
Treatment Treatment Incubation Final supplement concentration type number temperature (°C) (mg litre -I)
Killed control culture prepared for this treatment. b Resazurin (0-8 mg litre -1) plus sodium dithionite (32 g litre l) dissolved in 0.1m Tris-HCl buffer (pH 8.2).
184 P. Morgan, S. T. Lewis, R. J. Watkinson
During desorption the helium purge gas was passed through a Carlo Erba MFA515 cryogenic trap. This was cooled to -100°C with liquid nitrogen and served to con- centrate the desorbed BTEX in a short length of uncoated silica capillary just above the gas chromatograph column. At the end of the desorption period the trap was flash heated to 180°C in order to introduce the compounds to the gas chromatograph column. Gas chromatography was performed under the following conditions:
column: 30 m x 0.54 m m i.d. megabore column (J & W, part no. 1251334)
stationary phase: DB-624, 3-0/~m film thickness oven temperature: 35°C for 4 min then at 4°C min 1
to 190°C carrier gas: helium gas flow: 10 ml min injection: splitless from cryogenic trap detector: flame ionisation
Under these chromatographic conditions p- and m- xylenes co-migrated. Recovery efficiencies were deter- mined during each run by the use of appropria~te blanks and standards. Mean recoveries ranged from 85% for benzene to 95% for o-xylene. When dilution of water samples was necessary to prevent breakthrough from the trapping tubes this was performed using high purity water.
General analyses pH was determined using an Orion SA720 meter and electrode. Dissolved oxygen concentration was deter- mined using a W T W electrode. Elemental analysis was performed on filtered (0.45 /zm pore-size) samples by
means of a Jobin Yvon JY32 plasma emission spec- trometer. Nitrate, chloride and phosphate concentra- tions were determined in filtered samples by means of a Dionex 2000 series ion chromatograph fitted with an AS3 column using an eluent of 22 mM Na2CO3 - 28 mM N a H C O 3. Samples for total organic carbon mea- surement were acidified to pH <2 with phosphoric acid and sparged with helium gas for 1 h before analyis, in order to remove volatiles and carbonate. Analysis was performed on a Dohrmann DC80 instrument.
Total microbial cells were enumerated by acridine orange direct counts. Numbers of viable heterotrophic bacteria were determined by total viable counts on two media. General heterotroph were cultured on pep- tone-yeast extract-glucose agar. Bacteria capable of degrading volatile hydrocarbons were cultured on car- bon-free Bushnell-Haas mineral salts agar in the pres- ence of gasoline vapour. Colony counts were performed after 3 weeks incubation at 15°C.
RESULTS AND D I S C U S S I O N
Analysis of ground-water samples The analytical data for the samples collected from the three wells are given in Table 3. The experiments were performed at the ambient temperature of the field (15°C) except when an elevated temperature was specifi- cally required. All samples were aerobic but contained relatively low concentrations of oxygen (<2.5 mg kg 1). The samples were brown in colour. Since elemental analysis showed that the iron concentration of the water was below 7.0 mg litre ~, this colour was more probably due to humic compounds. The relatively high
Table 3. Properties of freshly collected ground-water samples
Total Pb (mg litre J) Total Mg (mg litre 1) Total Cu (mg litre i) Total Fe (mg litre i) Total P (mg litre 1) Total organic C (mg litre 1) Soluble CI (mg litre i) Soluble NO 3 (mg litre i) Soluble phosphate (mg litre 1)
<0.1 <0.1 <0.1 25-3 43.2 39-9
185 191 111 5.9 6.8 3-8
205 212 127 31 17 ND a
114 137 110 <0.5 <0.1 <0.1 0-5 <0.1 <0.1
AODC count (ml l) 5-6 x l0 s 8-4 × 10 5 1-3 × 10 6
Viable cell counts PYEG (cfu ml 1) 1.9 x 10 4 3.0 x l0 3 1.6 X 10 4
Gasoline (cfu ml i) <10 2 <10 2 <10 2
a ND--not determined.
Biodegradation in gas-condensate-contaminated ground water 185
Table 4. Extent of BTEX removal from ground-water sample from well 1 incubated under natural aerobic conditions for 13 days
l. Ambient 43" 70" 100 ~ 57 ~ 9 2. Ambient + 10°C 39" 92" 100 a 100 ~ 26" 3. Yeast extract 40 ~ 81 ~ 89" 100" 33" 4. N H a N O 3 19 83" 100 ~ 100" 0 5. Pi 30 68" 100" 52" 17" 6. NH4NO 3 + Pi (low) 44 a 74" 100 ~ 54" 24 ~ 7. N H a N O 3 + Pi (high) 48 ~ 92" 100 ~ 73" 39" 8. Urea+P i 38 ~ 78 ~ 84 ~ 68 ~ 23 a
Killed control 1 25 13 0 0 0 Killed control 2 28 7 0 0 0
"Elimination significantly greater than killed control treatments.
organic ca rbon content o f the water (up to 31 mg litre J) supports this hypothesis. The concentra t ion o f in- organic ni trogen and phosphorus ions was very low suggesting that inorganic nutrients may have been limiting microbial growth. However, the total phos- phorus concentra t ion was relatively high. In view o f the very low B T E X concentra t ion in the sample f rom well 3, no further studies were performed with this water. The concentra t ions o f all c o m p o u n d s except for benzene in water f rom well 2 were also low. This rendered determinat ion o f degradat ion difficult and all da ta for well 2 are for benzene only.
Biodegradation under natural aerobic conditions In samples o f ground-water incubated under natural aerobic conditions there was extensive removal o f BTEX by microbiological activity. In samples f rom well 1, it was possible to moni tor the removal o f all components with time. Table 4 lists the extent o f BTEX depletion as a function o f the different treatments performed. There was relatively little abiotic loss o f compounds except for benzene. This depletion may have been due to sorption onto the vial stoppers or to evaporative loss and may have been enhanced by the high initial concentrat ion o f this compound. Usually, BTEX elimination was signifi- cantly higher in biologically active treatments than in the killed controls. When this was not the case, there was no pattern in the distribution o f inactive treatments that would indicate that the treatments performed were in- hibitory. Figure 2 illustrates the concentrat ion o f BTEX components as a function o f incubation time in samples incubated under natural aerobic conditions. In these graphs killed control cultures are not illustrated for reasons o f clarity. There are no consistent significant differences between the treatments performed. Often, and particularly for benzene and toluene, there remained a stable residual concentrat ion o f contaminant following an initial period o f rapid biodegradation.
Owing to the very low concentra t ions o f toluene, ethylbenzene and xylenes in water f rom well 2, it was not possible to obtain reliable elimination data. The extent o f benzene elimination over the incubation period is given in Table 5. Degrada t ion was significantly
a) Benzene
20,000
18,000
16,000 ~ ==
14,000
12,000 CII
10,000
8,000 5 1'0 Time (days)
15
b) Toluene
1,800
1 , " 0 ~
A 1,4110
1,2oo "~ 1,000
"o ~. 600
400
200 "
O0 . . . . 5 . . . . 1'0 . . . . Time (days)
c) Ethylbenzene
200
~ " 15(
=o lOO
J >,
5 10 Time (days)
15
d) p- & m-xylenes
350
A 300
s. 250
2 . .
~, 150
E 1110 +
~" 50
0 O 5 10
Time (days) 15
e) o-xylene
8O0 q
rod
r'" 6o~
s "
4 . .
,~ 3 . .
2OO
100
oo 5 10 15 Time (days)
Fig. 2. Removal of BTEX from ground-water samples from well 1 under natural aerobic conditions as a function of different treatments. (*) Ambient conditions; (O) incubation at 25°C; (o) supplemented with yeast extract (18 mg litre-I); ( I ) supple- mented with NHaNO 3 (4.5 mg litre-l; (0) supplemented with phosphate buffer (pH 7; 2-2 mg litre i; (A) supplemented with NHaNO 3 (4.5 mg litre l) + phosphate buffer (pH 7; 2.2 mg litre i; (V) supplemented with NH4NO 3 (45 mg litre l) + phos- phate buffer (pH 7; 22 mg litre-i; (O) supplemented with urea (4.5 litre i + phosphate buffer (pH 7; 2-2 mg litre l). Data for
killed control cultures are not illustrated for reasons of clarity.
186 P. Morgan, S. T. Lewis, R. J. Watkinson
greater than that seen in the corresponding killed control cultures and there were no clear differences between the treatments performed. Usually benzene elimination was virtually complete. The elimination of benzene over the incubation period is illustrated in Fig. 3(a). Approximate rates of BTEX elimination were calculated from the slopes of plots in Figs 3 and 4 in order to illustrate potential differences between differ- ent treatments (see Table 7 below). In certain cases, the observed rates of BTEX elimination was clearly less for some treatments than for others. However, there was no consistent pattern to the results and therefore no one treatment was generally better or worse than the others. The rates obtained are comparable with those reported previously. Karlson and Frankenberger (1989) reported benzene and toluene degradation from initial concentrations of 400-600 /zg litre 1 in contaminated ground water at rates of approximately 305 /~g litre day ~ and 700/zg litre i day ~, respectively.
Despite the relatively low concentrations of oxygen in the ground-water samples, extensive biodegradation of BTEX was observed. The rates of degradation of all components were relatively rapid. In water from well 1, there remained a residual concentration of BTEX since biodegradation apparently ceased after several days in- cubation. Since elevated incubation temperature, the addition of organic nutrients or the addition of inor- ganic nutrients did not enhance the rate or extent of biodegradation, it is likely that oxygen depletion limited the quantity of contaminants that could be biodegraded. BTEX persistence as a function of oxygen deficiency has been widely reported (e.g. Barker et al., 1987; Chiang et al., 1987, 1989; Berwanger & Barker, 1988; Karlson & Frankenberger, 1989; Thomas et al., 1990). Direct correlation between the initial oxygen concentration in the water and the quantity of BTEX removed in the biologically active incubations was not possible. Assuming that the oxygen requirement for the conversion of BTEX compounds to biomass, carbon
Table 5. Extent of benzene removal from ground-water sample from well 2 incubated under natural aerobic conditions for 13
days or anaerobic denitrifying conditions for 85 days
Elimination significantly greater than killed control treatments.
dioxide and water is approximately 1.5 mg (mg sub- strate) ~ then the initial oxygen concentration of the water (2.1 mg litre ~) could have accounted for the removal of approximately 3200/xg BTEX litre ~. If metabolism of BTEX is to carbon dioxide and water without the production of new biomass, then the oxy- gen requirement per unit substrate is much higher. The quantity of BTEX removed by biodegradation during the incubation period ranged from approximately 2000 to 5000/zg litre ~, too high to be accurately correlated with the measured oxygen concentration. It may be that other electron acceptors were present in the water that lessened the oxygen demand for BTEX degradation. Alternatively, there may have been oxygenation of the water during the vial filling procedure. With water from well 2 degradation of BTEX was generally complete. Since the initial concentration of BTEX was below 500 /zg litre 1 and the oxygen concentration was 2.6 mg litre i there was sufficient oxygen to account for the biodegradation of all the contaminants present.
Elevating incubation temperature to 25°C did not
e) Aerobic
8 0 0 7OO
6OO
°°I 0 0 5 10 15
Time (days)
b) Denitrlfying
600
500 . ~
400 "7 v ; 300 ea
~ 200'
IO0
w
,o ,o 3'0 ' , ~ ~ 'o 6 ' 0 , ' 0 " ; o Time (days)
Fig. 3. Removal of benzene from ground-water samples from well 2 under natural aerobic and denitrifying conditions. (a) Standard aerobic conditions: (*) ambient conditions; (C)) incu- bation at 25°C; (×) supplemented with NH4NO3 (4.5 mg litrel; (0), supplemented with phosphate buffer (pH 7; 2.2 mg litre i; (A), supplemented with NH4NO 3 (4.5 mg litre -I + phosphate buffer (pH 7; 2.2 litre-l; (V) supplemented with NH4NO 3 (45 mg litre ~ + phosphate buffer (pH 7; 22 mg litre-I; (O) supplemented with urea (4.5 mg litre L) + phosphate buffer (pH 7:2.2 mg litre ~). Data for killed control cultures are not illustrated for reasons of clarity. (b) Denitrifying conditions: ((>) killed control; (A) standard denitrifying conditions; (V) supplemented with NH4NO 3 (4.5 mg liter l)+phosphate
buffer (pH 7; 2.2 mg litre-t).
Biodegradation in gas-condensate-contaminated ground water 187
Removal of BTEX from ground-water samples from under denitrifying conditions. (O) Killed control:
(A) standard denitrifying conditions; (V) supplemented with NH4NO3 (4.5 mg litre l) + phosphate buffer (pH 7;
2.2 mg litre i). There was no elimination of o-xylene.
stimulate the rate of degradation in these studies. It may be that the rate of degradation was limited by other environmental factors in the samples and that elevating the temperature was therefore unable to enhance metabolic rate. Alternatively, population changes may have occurred that were not reflected in increased incubation temperature or the experiment, as performed, may not have been able to resolve small alterations in rate.
Biodegradation under peroxide-supplemented conditions In order to try to enhance BTEX degradation in the ground-water samples, ground-water samples were supplemented with hydrogen peroxide to give a final concentration of 200 mg litre 1. It was found that this gave total inhibition of biodegradative activity in the ground-water from Uiterburen-- in no case was elimi- nation from the active cultures significantly greater than that seen in the killed controls. This can be attributed to toxicity of hydrogen peroxide to the microorganisms.
The enhancement of BTEX biodegradation in ground-water samples by oxygen supply has been demonstrated in both laboratory and field studies (e.g. Chiang et al., 1987, 1989; Karlson & Frankenberger, 1989; Thomas et al., 1990). However, oxygen transport is limited by the solubility of oxygen in water (Morgan
& Watkinson, 1989). For example, the solubility of oxygen in air-saturated water is 8-10 mg litre ~ and when pure oxygen is used this rises to 40-50 mg litre 1. Such quantities may not be sufficient to supply an ac- tively biodegrading aerobic population and conse- quently alternative oxygen supply techniques have been considered for use in aquifers. The most widely applied is hydrogen peroxide, since this compound decomposes to yield approximately equal amounts of oxygen and water. Some populations appear tolerant to hydrogen peroxide, for example, Brown et al. (1984) found that hydrogen peroxide concentrations as high as 400 mg litre i stimulated degradation in aquifer samples taken from a gasoline-contaminated site. Similarly, Hinchee and Downey (1988) reported no inhibitory effects on aquifer populations at a hydrogen peroxide concentra- tion of 300 mg litre ~. However, Huling et al. (1990) ob- served inhibition by hydrogen peroxide at con- centrations between 100 and 200 mg litre -]. These results demonstrate the site-specific nature of microbial tolerance of hydrogen peroxide. Since there may also be difficulties in using hydrogen peroxide, as a result of the chemical properties of the soil (Morgan & Watkinson, 1992), the deployment of this compound in bioremediation operations should be approached with caution.
Biodegradation under anaerobic conditions Under anaerobic conditions there was no significant removal of BTEX components in the absence of added nitrate. When nitrate was added, there was apparent biodegradation of benzene, toluene, ethylbenzene, and the p- and m-isomers of xylene. There was no degradation of o-xylene. Table 6 lists the extent of BTEX removal from water from well 1 over the 85-day incubation period. The equivalent data for benzene in water from well 2 are given in Table 5. The extent of degradation was consistently greater when inorganic nutrients were added to the water. This can be seen also from the plots of BTEX concentration against incubation time (Figs 3(b) and 4) and the observed approximate rates of degradation (Table 7). The rates of degradation under denitrifying conditions were very much slower than those obtained under aerobic conditions, but are comparable with those obtained by Hutchins et al. (1991a-c) and Gersberg et aL (1991). For example, under optimised conditions Hutchins et al. (1991a-c) obtained turnover rates of between 0.016 and 0.38 day l for toluene, ethylbenzene and xylenes present at initial concentrations between 3000 and 6000/xg litre 1. In this study, apparent turnover of benzene and toluene typically occurred at 0.01~0.04 day i.
The absence of biodegradation of BTEX in Uiterburen ground-water under anaerobic conditions when nitrate was not added confirms reports which found no evidence of anaerobic BTEX biodegradation (e.g. Battersby & Wilson, 1989) or only very slow degradation after a long lag period (Vogel & Grbic-Galic, 1986; Wilson et aL, 1986; Grbic-Galic & Vogel, 1987; Belier et al., 1991). Under denitrifying anaerobic conditions the
188 P. Morgan, S. T. Lewis, R. J. Watkinson
Table 6. Extent of BTEX removal from ground water sample from well 1 incubated under anaerobic denitrifying conditions for 85 days
17. Standard 2Y 68" 26 u 0 0 18. NH4NO3/P i 41 ~ 86 ~ 100" 44" 17
Killed control 17 11 21 1 12 22
Elimination significantly greater than killed control treatments.
response o f a microbial communi ty is site-specific. Previous reports have observed no degradat ion under denitrifying condit ions (Harr ison & Barker, 1987; Chiang et al., 1987, 1889; Berwanger & Barker, 1988), degradat ion o f some components (Berry-Spark & Barker, 1987; Evans et al., 1991a, b) and the degrada- t ion o f all c o m p o u n d s (Major et al., 1988; Gersberg et al., 1991). Al though observed in Uiterburen ground- water, the denitrifying degradat ion o f benzene has rarely been reported. Hutchins et al. (1991a-c) found that all BTEX componen t s were degraded under deni- trifying condi t ion in a contamina ted aquifer, but that trace concentra t ions o f oxygen in the ground-water could have accounted for the initiation o f metabolic at tack on the benzene, since this c o m p o u n d was not degraded under denitrifying condit ions in the labora- tory (Hutchins, 1991a, b).
There was a large excess o f nitrate relative to the BTEX concentra t ion added to the vials in order to ensure that any potential degradat ion was not nitrate limited. At tempts to relate the removal o f nitrate to the apparent biodegradat ion o f BTEX were not posi- ble, since complete nitrate removal occurred in all deni- trifying treatments (Fig. 5). This was probably due to the degradat ion o f other organic compounds in the ground-water (see Table 3) and relates to observations that have been made in the field at the trial bioremedia- tion operat ion at this site (Boks et al., 1990) and else- where (Hutchins et al., 1991c). At the site, nitrate was added to the soil in order to function as an electron acceptor for microbial respiration. However, there was no stimulation o f BTEX degradat ion, nor did any nitrate enter the aquifer, despite nitrate being added to the soil at concentrat ions up to 0.33 kg m 2. These
Table 7. Rates of BTEX removal from microbiologically active ground-water samples
1 120 N D c N D ND ND 2 250 ND ND ND ND 3 200 ND ND ND ND 4 180 ND ND ND ND 5 130 N D ND ND ND 6 86 ND ND ND ND 7 100 ND ND ND ND 8 90 ND ND ND ND
17 11 ND ND ND ND 18 9 ND ND ND ND
a Rate were calculated from linear portions of plots of compound concentration against time and are approximations only. Elimination of BTEX from killed controls was significantly slower (Tables 5 and 6). b Treatments 1-8 are under natural aerobic conditions and treatments 17 and 18 are under denitri- lying conditions. Details of the treatments are given in Table 2. c ND--not determined since initial compound concentration was too low to provide an accurate mea- sure of compound disappearance.
Biodegradation in gas-condensate-contaminated ground water 189
a) Wel l I
300
250 ~
200
150
o ,-:. :~ 100
5O
0 0 20 40 60
Time (days)
80 100
b) Wel l
300
2 0 0
200
1. ~ 150
1 ~ 100 z
so ~ ~
0 , ~ 20 40 60 80 100
Time (days)
Fig. 5. Nitrate depletion during incubation of ground- water samples under anaerobic denitrifying conditions. ( ~ ) Killed control; (A) standard denitrifying conditions; (V) supplemented with NH4NO 3 (4-5 mg litre ~) + phosphate
buffer (pH 7; 2.2 mg litre 1).
finding, together with those obtained in this report, would suggest that the added nitrate has been used by microorganisms degrading natural organic carbon rather than the BTEX contaminants.
C O N C L U S I O N S
It has been shown that BTEX can be degraded by natural microorganisms present in the ground-water at the Uiterburen site. All compounds were rapidly de- graded under natural aerobic conditions. Elevation of incubation temperature, supply of organic nutrients or addition of inorganic fertiliser did not increase the rate or extent of biodegradation and it appeared that the supply of oxygen was the factor limiting BTEX degra- dation at this site. Provided that the ground-water at this site remains aerobic, biodegradation of the BTEX contaminat ion present should occur and the process could be stimulated in a bioremediation operation of the type currently being tested (Boks et al., 1990). However, at tempts to increase the dissolved oxygen concentration in the ground-water by the addition of hydrogen peroxide to give a final concentration of 200 mg litre ~ completely inhibited biodegradation. Care to avoid toxic effects would therefore be necessary if
hydrogen peroxide were to be used in bioremediation at this site and similar responses may well be encoun- tered at other bioremediation locations.
No biodegradation occurred under anaerobic condi- tions, unless nitrate was provided as a terminal electron acceptor for microbial respiration. Under denitrifying conditions there was apparent biodegradation of benzene, toluene, ethylbenzene, m-xylene and p-xylene, but o-xylene was not biodegraded. Inorganic nutrient supplementation increased the degradation rate for samples from well 1. Degradation under denitrifying conditions occurred at a much slower rate than that under oxygenated conditions. The persistence of o- xylene and the slow degradation rate would appear to preclude bioremediation of BTEX-contaminated sites under denitrifying conditions. However, further work on this subject is justified to determine whether its application is practical.
A C K N O W L E D G E M E N T S
The authors would like to thank Nederlandse Aardolie Maatschappij B.V. (NAM) for supporting this work and, in particular, J. P. van Dessel for his encourage- ment and for organising access to the site. The authors also acknowledge the excellent field sampling performed on our behalf by IWACO B.V.
R E F E R E N C E S
Armstrong, A. Q, Hodson, R. E., Hwang, H.-M. & Lewis, D. L. (1991). Environmental factors affecting toluene degrada- tion in ground water at a hazardous waste site. Environ. Toxicol. Chem., 10, 147-58.
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