Article pubs.acs.org/est
Biodegradation of Chlorobenzene and Nitrobenzene at Interfaces between Sediment and Water Zohre Kurt, Kwanghee Shin, and Jim C. Spain* School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0512, United States S Supporting Information *
ABSTRACT: Plumes of contaminated groundwater often pass through an oxic/anoxic interface when they discharge into surface water bodies. We tested the hypothesis that contaminants recalcitrant under anaerobic conditions but degradable under aerobic conditions can be biodegraded at the interface resulting in the protection of the overlying water. Flow-through columns containing sediment and water were used to evaluate degradation of synthetic organic compounds at the thin organic layer at the sediment/water interface. Sediment samples collected from several sites contaminated with nitrobenzene (NB) or chlorobenzene (CB) were tested for their biodegradation capacities in the columns. The biodegradation capacities of sediment in the columns were 2−4.2 g CB·m−2·d−1 and 6.5 g NB·m2·d−1. Bacteria able to carry out rapid and complete biodegradation of CB or NB were detected in the sediments prior to the experiments, which suggested the presence of an active microbial community at the contaminated sites. The results revealed robust biodegradation of toxic compounds migrating across the sediment/water interface and indicate that the biodegradation capacities were sufficient to eliminate transport of the contaminants to the overlying water in the field.
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INTRODUCTION Much of the microbial activity in the biosphere takes place at interfaces that play key roles in a variety of ecosystems. Oxic/ anoxic interfaces are crucial features of all aquatic systems either in the water column or in the sediment. The interface can be meters thick in the water column1 whereas in sediments the interface usually varies from several millimeters up to several centimeters.2,3 In the water column, the position and depth of the interface is based on the oxygen production by phototrophs above and the flux of electron donors from below. In the sediment the position and depth is determined by many factors including temperature,4 molecular oxygen diffusion and advection to the sediment,4 microbial oxygen consumption, and sources and flux of electron donors.5 Methane, sulfide, hydrogen, and ferrous iron are important electron donors2,5−7 and oxygen, nitrate, ferric iron, and sulfate are relevant electron acceptors4 that support the growth of microbial communities. At oxic/anoxic interfaces the metabolic rates and the biomass of microbial communities in the sediment increase in proportion to the concentration of the limiting nutrient.8 The microbial communities are also responsible for the steep gradients of electron donors and acceptors at the interface and their composition and activities have recently been verified by clone libraries, DNA stable-isotope probing, and denaturing gradient gel electrophoresis.7,9−14 It is important to understand whether toxic organic compounds are able to behave like natural electron donors at © 2012 American Chemical Society
the sediment/water interface, because many anoxic contaminated groundwater plumes emerge in seeps or sediment and pass through oxic/anoxic interfaces.15,16 Several kinds of pollutants including nitroaromatic, chlorinated aliphatic, and chlorinated aromatic compounds can be converted under anoxic conditions to products that are aerobically biodegradable.17−19 While several studies have revealed biotransformation of haloaromatic compounds as they pass through organic sediment layers 20−22 most have focused on reductive dehalogenation in the anaerobic zones. Other studies report biodegradation of natural hydrocarbons from petroleum seeps as they emerge from the seafloor.23−25 The above interfaces also are analogous to the fringes of plumes in terms of being an intersection with steep redox gradients and high microbial activity.26−28 Despite the above studies, little is known about the aerobic degradation of synthetic organic compounds at the sediment/water interface. We designed simple laboratory columns to determine the potential of bacteria in sediments from two contaminated sites to degrade either nitrobenzene (NB) or chlorobenzene (CB). NB is made by nitration of benzene and is a major feedstock in aniline production. CB is produced by chlorination of benzene Received: Revised: Accepted: Published: 11829
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Table 1. Physical Properties, Potential Products of CB or NB Degradation, and Estimates of CB- or NB-Degrading Bacteria in Initial Sediment Samples sample RA-NB RB-NB RC-NB SA-CB SB-CB SC-CB SD-CB
pH 7.8 8.0 7.6 5.6 6.9 7.1 5.6
NO2− (μM) b
ND 2.8 ± 0.3 32 ± 3.4
NH4+ (μM)
Cl− (mM)
7.8 ± 0.5 32 ± 2.5 1.1 ± 0.2 1.2 0.52 0.76 1.2
± ± ± ±
MPNa/g
dry weight (mg of dry solids/mL of slurry)
0.057 0.023 0.019 0.057
11 17 32 340 53 100 303
± ± ± ± ± ± ±
0.4 0.8 1.1 11 5 9 13
3.7 4.0 2.4 1.4 1.6 4.0 1.4
× × × × × × ×
105 105 103 104 104 103 104
± ± ± ± ± ± ±
2.1 × 105 1.3 × 105 1.1 × 103 0.82 × 104 0.77 × 104 1.1 × 103 0.82 × 104
CB or NB was used as substrate for CB or NB MPNs respectively. Results are the means of eight replicates ±95% confidence intervals. bND means not detected.
a
prepared for each treatment. The degradation rates were determined based on the data after the lag period. Most Probable Number Analyses. Slurry samples were homogenized with either a tissue grinder with molecular grinding resin (Geno Tech. Inc., St. Louis, MO) or by brief (3 × 5 s) treatment in a bead beater (Biospec Mini Beadbeader) using 2.5 mm zirconia-silica beads prior to enumeration of bacteria by most-probable-number (MPN) estimation. Serial dilutions were prepared in 96-well microplates incubated in desiccators with either NB or CB vapors. The MPN was calculated from an 8-tube MPN table with 95% confidence limits.40 Cultures positive for CB degradation were indicated by a color change in bromothymol blue (20 mg/L) due to CB degradation and release of HCl.34 Cultures positive for NB degradation were indicated by turbidity increases quantified by measurement of A600 in a microplate reader. Analytical Methods. NB was analyzed as described previously with a Varian HPLC system equipped with a diode array detector.41 CB was analyzed using a Merck Chromolith RP18e column (4.6 × 100 mm) with a mobile phase that consisted of part A (0.1% trifluoroacetic acid (TFA) in water) and part B (0.05% TFA in acetonitrile) at flow rate of 1 mL/min. The mobile phase was changed from 100% part A to 100% part B over a 2-min period, and then held for 2 min. CB was monitored at 215 nm. Sediment samples were extracted with 1:1 acetonitrile/water prior to analysis. Oxygen was measured using a YSI model 58 oxygen probe. Chloride was determined based on the method of Yoshinaga using mercury thiocyanate.42 Dry weight, NO2−, NH4+, and pH of the sediments were quantified as described in Standard Methods.43 Column Design. Sediment samples were supported on Teflon frits (20-μm pore size) in glass columns (1.2 cm ID × 2.5 cm length) and the columns were sealed with Teflon stoppers (Figure 1). Stainless steel tubing (0.125 in. OD and 0.02 in. wall) was used for all connections to prevent sorption of NB or CB. Autoclaved sediments were used in the control columns for NB. Sterile controls for CB experiments were conducted after the active biodegradation phase by adding sodium azide (200 mg/L) to the influent. The feed solution was prepared with filter-sterilized site water or MSB supplemented with NB or CB at the indicated concentrations. Site water for NB studies was treated with activated carbon to remove unidentified organic compounds that interfered with HPLC analysis. The system was operated at 1 mL/h flow rate until biodegradation reached steady state after at least 10 hydraulic retention times, and then the flow was increased stepwise until breakthrough was observed. Column effluents were regularly collected in vials containing H2SO4 (1 mL of 0.5 N for NB and 2 mL of 1 N for CB) and analyzed by HPLC
and is used for manufacturing rubber, agricultural chemicals, antioxidants, dyes, and pigments. Both chemicals are contaminants in subsurface solids and groundwater at manufacturing sites. The biodegradation mechanisms for NB and CB under aerobic conditions are well established.29−36 Both compounds, however, seem to be relatively resistant to biodegradation in anoxic groundwater.37−39 The goal of our study was to test the hypothesis that biodegradation in a relatively narrow layer of oxic sediment could be sufficient to prevent migration of the contaminants to the overlying water.
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MATERIALS AND METHODS Chemicals. Acetonitrile, trifluoroacetic acid, CB, and NB were purchased from Sigma-Aldrich. 3-Chlorocatechol was obtained from Helix Biotech Corp. 14C-UL-NB was from Moravek with 99% radiopurity. Samples Used for the Study. Samples RA, RB, and RC were collected from the light organic layer at the surface of the sediment where a drainage ditch intersects an anoxic NB plume and in a wetland at a former chemical manufacturing site in New Jersey (Figure S1 in the Supporting Information). Similarly, samples SA, SB, SC, and SD were collected from the top of the sediment layer where an anoxic CB contaminated plume is intersected by a canal with a substantial amount of organic sediment at a former dye-manufacturing site in New Jersey (Figure S1). Physical and chemical parameters of all the samples are provided in Table 1. Microcosm Construction. Microcosms were constructed in serum bottles with sediment slurries and either Stanier’s mineral salts media (MSB)9 or filter- (0.2 μm) sterilized site water to determine the effect of inorganic nutrient addition. CB microcosms were constructed in 100-mL glass bottles with Teflon-lined stoppers and consisted of 1 mL of sediment slurry in 19 mL of media with CB. NB microcosms were constructed in glass tubes (200 mm × 25 mm) with Teflon-lined screw caps and consisted of 1 mL of sediment slurry in 19 mL of site water or BLK medium. NB was directly added to the liquid medium. Microcosms were incubated at room temperature. CB bottles were placed on a shaker at 200 rpm and NB tubes were placed on a Bellco roller drum operated at 17 rpm. Two active cultures and one autoclaved control were prepared for each treatment. Sediments were analyzed for dry weight, Cl−, NO2−, NH4+, and pH (Table 1). At appropriate intervals liquid samples from microcosms were analyzed by HPLC to determine NB or CB concentrations. Degradation Rate Determination. To determine the rates of degradation as a function of sediment concentration microcosms were prepared with 5−60 mg (dry weight) of sediment. Three active cultures and one killed control were 11830
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Figure 1. Schematic diagram of the column and continuous flow feeding system. Vial 1 contained 1 mL of 0.5 N H2SO4 (pH < 2); Vial 2 contained 10 mL of acidified 50% acetonitrile (pH < 2); Vial 3 contained 10 mL of 1.0 N NaOH (13 < pH < 14). Fractionation during radio tracer experiments is represented within the dashed rectangle.
(Figure 1). The second vial contained acidified 50% acetonitrile (10 mL) to capture volatilized nitrobenzene/chlorobenzene. For radiolabeled experiments a third trap contained 1.0 N NaOH. The vials were sparged with air for 1 min when the trapped samples were removed for analysis. The degradation rates were determined when the columns reached each new steady state. Mineralization in Columns. For NB columns filtersterilized site water supplemented with unlabeled (6.15 mg/ L) and 14C-ring labeled NB (4.5 μCi/L) was fed for 2 h at a flow rate of 2 mL/h into the bottom of the columns via a syringe pump. Samples of column effluents were fractionated into aqueous, volatile organic compounds, biomass, and 14CO2 (Figure 1). Each fraction was mixed with scintillation cocktail and analyzed by liquid scintillation counting (LSC). The columns were sacrificed at the end of the experiments for mass balance analysis. CB mineralization was estimated by measuring chloride and oxygen in the feed and effluent of the column.
Figure 2. CB biodegradation and chloride release in microcosms prepared with sediment SA using site water (A) or MSB (B): black ▲, active cultures (duplicates); black ●, killed control; blue ▲, chloride in active cultures (duplicates); blue ●, chloride in killed control.
The resulting degradation rates were 0.36 ± 0.06, 3.7 ± 0.4, 1.6 ± 0.2, and 0.49 ± 0.05 μg of CB·mg of sediment−1·d−1 for SA, SB, SC, and SD samples, respectively. The depths of sample collections were not well controlled and although MPNs indicate substantial populations of CB degraders, the experiments were not designed to evaluate in situ CB degradation rates or populations in the most active zones. Thus the rates are conservative estimates of the potential for degradation in the field. The presence of substantial populations of cultivated CBdegrading bacteria and the rapid degradation in microcosms supports the hypothesis that CB degradation is taking place in the field where the contaminant plume encounters oxygen at the sediment/water interface. MSB did not enhance the degradation of CB, which indicated that the overlying water in the canal contained sufficient inorganic nutrients to support the biodegradation process. Stoichiometry of Oxygen Demand during Degradation. Oxygen serves as the electron acceptor during mineralization of CB, thus degradation at the sediment/water interface in the field strictly depends on the availability of oxygen from the overlying water because the plume is anoxic. When ultimate biochemical oxygen demand (BOD) was measured for CB using the sediment samples as seed,43 9.2 mg of oxygen·L−1 were required to degrade 10 mg of CB·L−1. The BOD was similar to the oxygen demand calculated when 50% of the compound was converted to biomass. In column studies described below oxygen and CB concentrations in the feed
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RESULTS AND DISCUSSION Initial characterization of sediment samples (Table 1) provided evidence of possible natural attenuation in the field. Neither CB nor NB was detected in any of the samples but bacteria able to degrade the compounds of interest were present. High chloride concentration and low pH in some samples could indicate ongoing or recent in situ CB biodegradation.34 Similarly the likely byproducts of the NB degradation pathway, nitrite and ammonia, quantified in the slurry could support previous investigations at the chemical manufacturing site that indicated ongoing NB biodegradation.44,45 Chlorobenzene Biodegradation. Microcosms. Because of the substantial populations of bacteria able to use CB as a growth substrate in the surficial sediment at the contaminated site (Table 1), degradation of CB was rapid after brief lag periods in all treatments except abiotic controls (Figure 2 and Figures S2−S3). A second addition of CB resulted in its immediate and rapid disappearance, which clearly indicated biodegradation leading to acclimation of the microbial population. Total numbers of CB degraders increased dramatically upon enrichment in the microcosms (Table S1). MPN results correlated well with chloride production and CB disappearance, providing clear evidence for CB mineralization by the microbial community in the sediment. 11831
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of sediment−1·d−1 at a flow rate of 2 mL·h−1, which indicated that a 2-mm active zone at the sediment/water interface would have a CB degradation capacity of 2.0 ± 0.3 g·m−2·d−1 at a flow of 424 L·m−2·d−1. The results demonstrated clearly that a very thin sediment layer representative of the sediment/water interface in the canal has a high capacity for CB degradation. Mass Balance of CB. The CB mass balance was established based on chloride measurements and indicates that as expected one mole (0.85−1.15) of chloride was produced per mole of CB degraded (Figure 3 and Figures S4−S6)34,35 Taken with the growth of the CB degrading populations in microcosms the close correspondence between chloride released and CB disappearance indicates that the CB was completely degraded. The results established that the disappearance of CB was due to biodegradation and the mass balance was within 20% of theoretical throughout. Nitrobenzene Biodegradation. Most Probable Number Analysis. MPN analyses indicated that, although populations vary among sample sites, there were significant numbers of cultivated bacteria able to use NB as growth substrate in the surficial sediment at each of the sites (Table 1). Microcosms. After a brief acclimation period NB degraded rapidly in all sediment amended microcosms except the killed controls (Figure 4 and Figure S7). Degradation was immediate
were adjusted to ensure that excess oxygen was available (Figure S5). Chlorobenzene Column Studies. Continuous flow column systems were designed to determine the potential for the aerobic microbial community at the sediment/water interface to degrade contaminants as they transit the interface. For contaminants that are degraded predominantly by aerobic mechanisms, oxygen is the limiting factor in contaminant plumes emerging into aerobic receiving waters. For this study the goal was to eliminate the influence of oxygen mass transfer and determine the metabolic potential of the bacteria. Therefore, oxygen was provided in the filter-sterilized feed solution along with the contaminants. In the field oxygen would be supplied by diffusion from the overlying water. Column experiments for determination of CB degradation were conducted with each of the 4 sediment samples (Figure S1A) from the canal (Figure 3, Figures S4 and S6). CB (88
Figure 3. CB biodegradation in column containing SD sediment (dry weight of 606 ± 22 mg at a height of 2 mm): black ▲, CB in effluent; ●, CB in feed; blue ▲, chloride release; red ---, flow rate. Figure 4. Biodegradation of NB with site water and RA sediment (dry weight of 17.1 ± 0.8 mg): ▲, active cultures (duplicates); ●, killed control.
μM) was completely degraded at feed flow rates from 1 to 2 mL·h−1 with sediment sample SB and SC (Figure S4). When flow rates were increased to 5 mL·h−1, CB appeared in the effluent and then declined within 30 h until the experiment was terminated. Degradation rates would be expected to increase with longer acclimation periods at the highest flow rates. At a flow rate of 2 mL·h−1 with sediment heights of 2 and 3 mm the biodegradation capacities of the columns were 4.5 ± 0.8 μg of CB·mg of sediment−1·d−1 and 2.3 ± 0.3 μg of CB·mg of sediment−1·d−1 for SB and SC, respectively. The capacity of the 2-mm sediment/water interface was, therefore, 4.2 ± 0.5 g·m−2·d−1 at a flow of 424 L·m−2·d−1. CB (41 μM) was completely degraded at flow rates from 1 to 4 mL·h−1 in the column containing SA sediment. When flow rates were increased to 6 mL·h−1, channeling artifacts appeared, therefore, the degradation rates were calculated based on the 4 mL·h−1 flow rate. The capacity of the SA sediment layer column (2 mm) was 0.6 ± 0.05 μg of CB·mg of sediment−1·d−1 at a flow of 4 mL·h−1 and the degradation capacity of the sediment layer was therefore 3.9 ± 0.4 g·m−2·d−1 at a flow of 848 L·m−2·d−1. SD sediment degraded CB at flow rates up to 2 mL·h−1 and channeling happened at 3 mL·h−1 (Figure 3). The capacity of the SD sediment layer was 0.4 ± 0.02 μg of CB·mg
after a second addition of NB, which clearly indicated that NB disappearance was due to biodegradation by the acclimated microbial community. The degradation rate constants were 15 ± 1, 2.5 ± 0.4, and 5.6 ± 0.8 μg of NB·mg of sediment−1·d−1 of sediment in samples RA, RB, and RC. The relatively short acclimation period and facile isolation of NB degraders suggested that bacteria were actively degrading the NB in situ. Because the microbial biomass in the field is proportional to the flow of the limiting nutrient, the differences in initial biomass and degradation rates probably reflect different rates of contaminant flux at the three sampling locations. Stoichiometry of Oxygen Demand during NB Degradation. Because it is degraded using oxygen as the electron acceptor,46,47 NB degradation at the sediment/water interface in the field depends on the availability of oxygen from the overlying water. The stoichiometry of NB biodegradation was previously estimated as 2.32 mols of oxygen per mole of NB based on respirometry with resting cells.47 Based on that estimate 3.7 mg of oxygen would be required for the 11832
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Table 2. Mass Balance of 14C-Labeled Nitrobenzene in Column (% of Initial Radioactivitya) column effluent active column sterile column a
sediment
CO2
aqueous
cells particulate
total
aqueous
bound
total recovery
58.4 ± 3.5 0.4
5.3 ± 0.4 89.4
1.8 ± 0.1 1.7
18.5 ± 0.6 0.6
0.3 ± 0.1 0.4
17.3 ± 0.6 0.6
85.8 91.4
Duplicate samples.
detritus mineralize NB as it flows through the sediment/water interface. Our study demonstrated clearly that the bacteria at sediment/water interfaces have a remarkable capacity to biodegrade contaminants as they migrate from a subsurface plume into the overlying water within a thin layer (2−3 mm) of sediment. The rates determined here were site specific and might be higher than in the field because oxygen concentrations were higher than would be expected in the lower portion of the oxic/anoxic interface. On the other hand, in the field the oxygen concentration at the top of the oxic/anoxic interface could well be saturating or even higher if phototrophs are present. Where gradients of electron donors and acceptors intersect, bacterial biomass can be highly concentrated in a thin layer. For example, in microbial mats48 or in ponds producing sulfide or methane that rise to meet oxygen produced by phototrophs,49 the chemolithotrophs or methanotrophs increase in number until the electron donor or acceptor becomes limiting. In laboratory columns the microbial communities responded rapidly and adjusted to take advantage of the increased availability of carbon and energy50 when the flux of the contaminants increased. The conclusion was supported by MPN increases in the microcosms (Table S1). The population change was not determined in the columns because the same columns were used as killed controls, however, when the flux of the contaminants increased, the microbial communities responded rapidly and adjusted to take advantage of the increased availability of carbon and energy.50 Bacteria able to degrade CB and NB in the columns were isolated and identified. Pseudomonas pseudoalcaligenes, Stenotrophomonas maltophilia, and Achromobacter piechaudii strains were isolated by selective enrichment with CB from the column sediments. Rapid consumption of 3-chlorocatechol and release of chloride in cell extracts treated with 0.01% hydrogen peroxide33,34,51 indicated that the isolated strains were using the modified ortho pathway for CB biodegradation. Pseudomonas sp, P. mendocina, and P. putida strains were isolated from NB columns by selective enrichment with NB. During growth on NB the release of ammonia rather than nitrite indicated that the isolates used the partially reductive NB degradation pathway.46,47 We assumed that the observed biodegradation took place in the thin sediment layer, but some biodegradation could be taking place in the water layer above the sediment, which never exceeded 16 mm in depth. In the field the interface might migrate vertically, but there will always be an interface where oxygen becomes available as the plume discharges to surface water. Sufficient oxygen was provided to the columns described here to prevent electron acceptor limitations at the sediment/ water interface. The goal was to determine the degradation capacity of the system when mass transfers of electron donors and acceptors were not limiting. Processes such as anaerobic oxidation of methane and sulfate reduction at the sediment and sediment/water interface have been modeled based on geochemical field data.52 Similarly,
degradation of 6.1 mg of NB. The oxygen demand of the NB used in the column experiments was, therefore, well below the solubility of oxygen in the feed and would be sufficient even if a slight increase of oxygen demand due to nitrification of ammonia is considered. The degradation of NB was verified by isotope analysis (Table 2) which demonstrated that NB disappearance was due to mineralization. Nitrobenzene Column Studies. In columns containing sediment from the NB contaminated site NB (50 μM) was completely eliminated at flow rates from 1 to 5 mL·h−1. When flow rates were increased to 10 mL·h−1 NB appeared in the column effluents and then declined over 3 h as the biomass became acclimated (Figure 5). The NB degradation rate was
Figure 5. Effluent concentration of nitrobenzene in a continuous flow column. Influent concentration was 50 μM. Columns contained a 2mm layer of sediment (dry weight of 58.0 ± 1.2 mg). Sediments used to construct the column were taken from the NB microcosms: ●, active column; ▲, sterile column; red ---, flow rate.
12.7 ± 1 μg of NB·mg of sediment−1·d−1 calculated at a flow rate of 5 mL·h−1. The results indicated a remarkably high capacity of the sediment based on a depth of 2 mm and flow rate of 1061 L·m−2·d−1). Mass Balance for NB. To determine the mass balance and the fate of NB as it flowed through the sediment/water interface, a pulse of 14C uniformly labeled NB was supplied to columns. A total of 58% of the radioactivity was recovered as carbon dioxide in the traps of the active columns at the end of the experiment. An additional 19% of the radioactivity was associated with cells or bound to sediment (Table 2). The sediment-bound radioactivity was attributed to radioactivity incorporated in biomass because negligible radioactivity was recovered from sediment in the control column. No other radioactive products were identified by HPLC. The results indicate clearly that indigenous bacteria associated with organic 11833
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laboratory data can also be used in mathematical modeling of biodegradation to predict whether biodegradation will be sufficient to lower contaminant concentrations to acceptable levels.53,54 The column approach here was system specific and designed to determine the capacity of the microbial community in a narrow sediment/water interface to degrade contaminants and protect the overlying water. The contaminant concentrations were consistent with concentrations at the field sites but the rates are site specific and no attempt was made to include a wide range of conditions. The principle that the microbial community will acclimate to completely degrade the limiting nutrient is well established in other biological systems. Our results indicate that the principle applies to sediment/ water interfaces where contaminated plumes emerge into surface water and the conclusion can be extrapolated to other sediment/water interfaces.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
S Supporting Information *
Additional tables and figures as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
Corresponding Author
*Phone: (404) 894-0628; fax: (404) 894-8266; e-mail: jspain@ ce.gatech.edu. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Funding was provided by DuPont Corporate Remediation Group. We thank the sampling team from URS for collecting the samples. We also thank E. Erin Mack and Shirley Nishino for reviewing the manuscript and help throughout the study.
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