APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2006, p. 449–456 0099-2240/06/$08.00⫹0 doi:10.1128/AEM.72.1.449–456.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 1
Effects of Endogenous Substrates on Adaptation of Anaerobic Microbial Communities to 3-Chlorobenzoate Jennifer G. Becker,* Gina Berardesco,† Bruce E. Rittmann,‡ and David A. Stahl§ Department of Civil and Environmental Engineering, Northwestern University, Evanston, Illinois 60208-3109 Received 21 June 2005/Accepted 17 October 2005
Lengthy adaptation periods in laboratory studies evaluating the potential for contaminant biodegradation in natural or engineered environments may indicate that the native microbial communities are not metabolizing the contaminants in situ. In this study, we characterized the adaptation period preceding the biodegradation of 3-chlorobenzoate in anaerobic communities derived from lake sediment and wastewater sludge digesters. The importance of alternative mechanisms of adaptation of the anaerobic communities to 3-chlorobenzoate was evaluated by monitoring the concentrations of metabolic substrates and products as well as the levels of total small subunit (SSU) rRNA and SSU rRNA from populations thought to be important in 3-chlorobenzoate mineralization. The anaerobic environments from which the 3-chlorobenzoate-degrading communities were derived contained different levels of endogenous substrates. Increasing methane levels in the digester and sediment communities and decreasing chemical oxygen demand concentrations in the sediment community during the adaptation periods revealed that endogenous substrates were preferentially utilized relative to 3-chlorobenzoate. Methane and chemical oxygen demand concentrations leveled off concomitantly with the onset of 3-chlorobenzoate biodegradation, suggesting that depletion of the preferentially degraded endogenous substrates stimulated 3-chlorobenzoate metabolism. Consistent with these observations, adaptation to 3-chlorobenzoate occurred more rapidly in digester samples that were depleted of endogenous substrates compared to samples that contained high levels of these biodegradable compounds. Other potential adaptation mechanisms, e.g., genetic change or selective population enrichment, appeared to be less important based on the reproducibility and relative lengths of the adaptation events, trends in the SSU rRNA levels, and/or amplification of SSU rRNA genes from key populations. the potential for exposure of humans and other organisms to contaminants. Clearly, the persistence and potential impact of some compounds in natural and engineered environments may be controlled to a greater extent by the adaptation period than by the rate of biodegradation following adaptation. Despite its importance, current understanding of the events that lead to microbial community adaptation to pollutants is extremely limited. This lack of understanding means that we are unable to predict when or where adaptive events will occur. Consequently, most studies of xenobiotic biodegradation instead focus on characterizing the biotransformation phase that follows the adaptation period under conditions of rapid pollutant removal. Key measurements that would make it possible to evaluate the importance of alternative adaptation mechanisms are generally not made during the adaptation period. In this study, anaerobic microbial communities derived from freshwater lake sediment and municipal wastewater sludge digesters and exposed to 3-chlorobenzoate (3-CB) were used as model systems for studying adaptation. Like many other halogenated aromatics, 3-CB has previously been shown to undergo biotransformation in anaerobic sediment (18, 25) and digester communities (20) but often only after extended adaptation periods. In most complex ecosystems, including the anaerobic sediment and digesters used as sources of inocula in this study, microorganisms are presented with myriad substrates (23). Utilization of endogenous substrates, compounds that are present in a culture inoculum and readily metabolized by the culture, may influence the biodegradation of contaminants in several different ways. Endogenous substrates may have little
Laboratory studies are frequently used to assess the potential for biodegradation of contaminants by the native microbial communities in natural and engineered environments. In many cases, biodegradation is eventually observed, but for a wide variety of organic compounds it is frequently preceded by a lengthy adaptation period, during which significant reductions in the concentration of the compound of interest do not occur (43). This is especially true of anaerobic biodegradation studies conducted with benzene and alkylbenzenes (16, 22, 31), fuel oxygenates (14, 38, 44), hexadecane (45), chlorinated phenols (7, 18, 36), chlorinated benzoates (25), and a wide variety of other contaminants. Long adaptation periods in laboratory biodegradation studies are significant, because they may indicate that indigenous microorganisms are not adapted to the contaminant(s) and may not be metabolizing these compounds at a detectable rate in situ (35). Further, if initially persistent pollutants are relatively mobile in groundwater aquifers or are introduced to a continuous-flow treatment system, contaminants may be washed out of an aquifer or treatment system before the microbial community can degrade them. This in turn increases * Corresponding author. Present address: Department of Biological Resources Engineering, University of Maryland, College Park, MD 20742-2315. Phone: (301) 405-1179. Fax: (301) 314-9023. E-mail:
[email protected]. † Present address: zuChem, Inc., Chicago, IL 60610. ‡ Present address: Center for Environmental Biotechnology, Biodesign Institute at Arizona State University, Tempe, AZ 85287-5701. § Present address: Department of Civil and Environmental Engineering, University of Washington, Seattle, WA 98195. 449
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effect if the contaminant and low concentrations of endogenous substrates are utilized simultaneously. Metabolism of higher concentrations of endogenous substrates may accelerate contaminant biodegradation if the growth of key organisms is stimulated by the utilization of the endogenous substrates. Alternatively, contaminant biodegradation may be delayed by the preferential utilization of endogenous substrates. The goal of this study was to examine how the metabolism of endogenous substrates and 3-CB were related and the relative importance of different potential adaptation mechanisms in the sediment and digester cultures following exposure to 3-CB. MATERIALS AND METHODS Source of inoculum. The adaptation experiments were conducted using an anaerobic sediment or digester sludge inoculum. Sediment was collected from the previously described (26) Fox Point sampling site in Lake Michigan using a box corer, transferred to canning jars, and stored at 4°C for approximately 3 months until used. Digester sludge was obtained from the North Shore Sanitary District’s Clavey Road Wastewater Treatment Plant in Highland Park, Illinois. After some material was flushed from a sampling line that contained primary and secondary digester tank contents, canning jars were filled until they overflowed and were stored at 4°C until used (within 24 h). Establishment of cultures and controls. Strict anaerobic and aseptic techniques based on the methods described by Miller and Wolin (27) were used throughout the study. Anaerobic slurries comprising sediment or digester sludge inoculum and anaerobic mineral medium were established using the methods described by Becker et al. (6). Two slightly different anaerobic mineral media formulations were used for the adaptation experiments. The medium utilized in the sediment adaptation experiment has been previously described (15). The medium used to dilute the digester sludge inoculum was similar to that used in the sediment adaptation experiment and contained the following compounds (in grams/liter): NH4Cl, 0.5; K2HPO4, 0.4; MgCl2 · 6H2O, 0.1; CaCl2 · 2H2O, 0.1; Na2S · 9H2O, 0.5; NaHCO3, 4.0; and yeast extract, 0.05. It was supplemented with resazurin and trace metal solution (10 ml) that was modified from the methods of Tanner (41) by adding H3BO4 (0.019 g/liter) and adjusting the pH of the nitrilotriacetic acid solution to 6.5 using NaOH. 3-CB (ⱖ99% pure; Aldrich Chemical Co., Milwaukee, WI) was added to give an initial concentration of 200 M. For each adaptation experiment, the following cultures and controls were maintained: a culture amended with 3-CB (2 liters and two or three 160-ml cultures), a live control with no 3-CB added (2 liters), and a 3-CB-amended sterile control (2 liters). Sterile controls were prepared by autoclaving the inocula for 1 h on three consecutive days before combining them with sterile medium and 3-CB. All cultures and controls were maintained with a headspace of 70% N2–30% CO2 and incubated statically at 30°C, except during sampling events. Adaptation experiments. Immediately following the first exposure of the amended sediment and digester cultures to 3-CB, all of the live cultures and controls were initially sampled for chemical and molecular analyses, as detailed below. Cultures were subsequently sampled at approximately 1-week intervals during the adaptation period, transformation of the initial 3-CB addition, and transformation of a second addition of 3-CB for a total of approximately 130 days. During sampling events, which lasted for several hours, the cultures were continuously shaken on a platform shaker (100 rpm, 30°C). 3-CB concentrations were monitored in all 3-CB-amended cultures and controls. Total and population small-subunit rRNA (SSU rRNA) levels were quantified in all live cultures. Community DNA was also routinely extracted from samples of the live cultures. For the sake of brevity, the results of the rRNA and DNA analyses are reported only for the 2-liter cultures. All other analyses were performed only on the live 2-liter cultures and controls. Dissolved chemical oxygen demand (COD) was monitored in the sediment cultures but not in the digester cultures. Endogenous substrate depletion experiments. 3-CB removal was also monitored in digester cultures that had previously been depleted of endogenous substrates. The live control used in the adaptation experiment was the source of the substrate-depleted culture. It was maintained in the absence of added growth substrates for approximately 6 months before duplicate 50-ml aliquots were aseptically and anaerobically administered to 160-ml serum bottles. These aliquots were equilibrated with 70% N2–30% CO2, sealed, incubated for 4 weeks, and then dosed with 3-CB, as described above for the adaptation experiments. Duplicate aliquots of the 3-CB-degrading digester culture were dispensed at the same time as those derived from the substrate-depleted live control. These 3-CB-degrading cultures were resupplied with 3-CB within 2 weeks of being
APPL. ENVIRON. MICROBIOL. transferred and served as adapted, endogenous substrate-depleted controls. The two pairs of cultures were periodically sampled for analysis of 3-CB. Analytical methods. The aqueous concentrations of 3-CB were determined using reverse-phase high performance liquid chromatography (HPLC) with diode array detection as previously described (6). Headspace CH4 and H2 concentrations were determined by gas chromatography with flame ionization and reduction gas detectors as previously described (5). COD measurements were made using a Hach method (Method 8000; 1 150 mg/liter COD) (17) as modified by Becker et al. (5). RNA extraction and quantification. The total small-subunit rRNA (SSU rRNA) concentrations in the sediment and digester cultures were quantified by hybridization to a universal oligonucleotide probe (S-*-Univ-1392-a-A-15), which targets virtually all known life (46). SSU rRNA in the sediment and digester communities was also hybridized with the Desulfomonile tiedjei-specific probes S-S-Dtied-1032-a-A-22 (GAA GAG GAT CGT GTT TCC ACG A [13]) and S-S-Dtied-0617-a-A-23 (TCG AAT GCA CTT CCG AGG TTG AG [this study]), respectively. The Archaea-specific probe S-D-Arch-0915-a-A-20 (2) was used to quantify methanogens in the digester and sediment cultures and controls (4, 5). Members of the genus Syntrophus were quantified in these systems using probe S-G-Syn-0424-a-A-18 (5). RNA was extracted from 3-ml (digester) or 4-ml (sediment) slurry samples using the mechanical disruption/phenol-chloroform extraction process of Stahl et al. (39) with previously described modifications (5, 26). RNA slot blotting, probe labeling, prehybridization, and washing were performed as previously described (34). Samples were blotted on nylon membranes in triplicate, prehybridized at 40°C, and washed at the experimentally determined temperature of dissociation. Temperatures of dissociation for the D. tiedjeispecific probes (48°C, S-S-Dtied-1032-a-A-22; and 58°C, S-S-Dtied-0617-a-A-23) were determined using a previously described elution method (34). The hybridized membranes were exposed to Storage Phosphor Screens (Molecular Dynamics, Sunnyvale, CA), which were scanned with a PhosphorImager (Molecular Dynamics). The resulting digitized images were analyzed using ImageQuant software (Molecular Dynamics). The concentrations of total SSU rRNA in the slurry samples were determined by comparison with a dilution series of reference RNA (from Escherichia coli). DNA extraction, amplification, separation, and sequencing. At each sampling interval, DNA was isolated from the 3-CB-degrading sediment and digester cultures and the associated live controls using the freeze-thaw and lysozyme treatments, phenol-chloroform extraction, and ethanol precipitation of Tsai and Olson (42) with previously described modifications (4). DNA obtained at several sampling events during the course of the adaptation experiments was amplified using Bacteria-specific primers and PCR and was separated using denaturing gradient gel electrophoresis, as described by Muyzer et al. (29) and modified by Becker et al. (4). Bands of interest in the denaturing gradient gel electrophoresis gels were excised and reamplified prior to sequencing (4). In addition, DNA obtained from the 3-CB-amended digester culture and control was amplified using the Desulfomonile tiedjei-specific primers S-S-Dtied-0059-a-S-22 (CAA GTC GTA CGA GAA ACA TAT C [13]) and S-S-Dtied-1032-a-A-22. These primers were reportedly used to detect D. tiedjei cells at a concentration of approximately 106 cells per g of nonsterile soil (13). The identity of PCR products was confirmed through sequencing and analysis as previously described (4).
RESULTS Adaptation experiments. To evaluate the potential importance of initial conditions or random changes (e.g., mutations) in the adaptation of the sediment and digester cultures, the reproducibility of adaptation to 3-CB was evaluated in replicate cultures. The lengths of the adaptation periods reported here reflect the day on which the concentration of 3-CB decreased 5% or more compared to the average of all previous measurements. Although adaptation to 3-CB in the sediment and digester cultures was a lengthy process, it was reproducible. In the sediment culture, biotransformation of the initial 3-CB dose was observed within 77 to 83 days, and removal of a second addition of 3-CB on day 107 or 114 began immediately (Fig. 1A). Similarly, biotransformation of 3-CB was initially observed in the digester community within 53 to 75 days, and a second spike of 3-CB, administered on day 95, was transformed in the four 3-CB-amended vessels without delay
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(Fig. 2A). Adaptation periods of similar durations were observed in cultures inoculated with sediment or digester sludge collected at various times throughout the year and stored for different lengths of time (data not shown). Benzoate was presumably produced but did not accumulate to detectable levels in the 3-CB-degrading cultures. No significant removal of 3-CB was observed in the sterile controls for over 125 days. H2 is an important intermediate in the mineralization of organic compounds under anaerobic conditions. Many H2-producing reactions, as well as H2-consuming processes, including reductive dehalogenation, may be kinetically and/or thermodynamically controlled by the amounts of H2 that accumulate. Therefore, the headspace H2 concentrations in equilibrium with aqueous H2 in the sediment and digester cultures were monitored to assess the potential role of H2 in controlling metabolism of 3-CB. Relatively high headspace H2 levels (⬎100 ppm to ⬃1,000 ppm) were initially measured in all 3-CB-degrading cultures and controls (Fig. 1B and 2B). However, by day 28, headspace H2 concentrations measured in the sediment culture and control had dropped to between 10 and 30 ppm, and they remained at this level during the remainder of the adaptation period and throughout the 3-CB biodegradation phase. Similarly, headspace H2 levels measured in the digester culture and control dropped to ⬃15 ppm within several days of adding 3-CB and remained low for the remainder of the experiment. During the adaptation period preceding 3-CB metabolism, the cumulative amounts of methane produced in the sediment culture and control were very similar and increased exponentially (Fig. 1C). By the time significant transformation of 3-CB was first noted in the sediment culture (day 77), endogenous methane production was over 80% complete (based on the total amount of CH4 produced in the control during the entire experiment). After the onset of 3-CB biodegradation, the differences in the methane levels in the 3-CB-degrading sediment culture and control became increasingly larger. During this time, endogenous substrate utilization tapered off in both systems, and increases in methane levels in the 3-CB degrading culture after day 77 were nearly equal to the methane production due to the utilization of 3-CB according to following equation: C6H4ClCOO⫺ ⫹ 8.5 H2O 3 3.5 CH4 ⫹ 3.5 H⫹ ⫹ 3.5 HCO3⫺ ⫹ Cl⫺. CH4 production totaled 605 mol and 1,495 mol during the two periods of 3-CB transformation, compared to 677 mol and 1,595 mol of CH4 expected based on the reaction stoichiometry in the above equation. Not surprisingly, CH4 production in the 3-CB-degrading digester culture and control (Fig. 2C) was much higher than in their sediment counterparts because of the higher organic matter content of digester sludge compared with the lake sedi-
FIG. 1. Characterization of an anaerobic lake sediment culture during adaptation to and transformation of 3-chlorobenzoate (3-CB). (A) 3-CB in a single 2-liter culture (filled circles), duplicate 160-ml cultures (open diamonds and circles), and a single 2-liter sterile control (filled triangles); (B) headspace H2; (C) CH4; (D) chemical oxygen demand (COD); and (E) total SSU rRNA. (B to E) Each datum point represents the concentration in a single 3-CB-amended 2-liter culture (filled circles) or unamended live control (open triangles).
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ment. However, CH4 production followed similar trends in the sediment and digester systems. Significant transformation of 3-CB in the amended digester culture was first detected on day 53. At the same time, CH4 production in this culture and the control began to level off. By day 53, over 80% of the total amount of CH4 generated in the no-substrate control over the course of the 126-day experiment had been produced in the control and 3-CB-degrading culture. In the 3-CB-degrading culture, CH4 production in excess of that generated in the control after day 53 represented ⱖ93% of the amount that would be produced from 3-CB mineralization according to the above equation. Soluble COD concentrations were measured in the 3-CBamended sediment culture and control (Fig. 1D). The COD concentrations provided an aggregate measure of the predominantly organic oxidizable matter in the sediment and are related to the abundance of biodegradable substrates (3). The difference in the initial COD concentrations in the 3-CB-degrading sediment culture and control represents approximately 87% of the theoretical oxygen demand associated with the initial dose of 3-CB in the amended culture (48 mg/liter O2). COD concentrations increased in the culture and control on days 16 and 28 following the addition of NaOH to adjust the pH on days 12 and 23. It is possible that the addition of NaOH increased soluble COD concentrations by altering the extent of substrate sorption onto soil particles or by lysing cells and releasing soluble, oxidizable cell constituents. Between days 28 and 70, the COD levels in the sediment culture and control declined approximately 41 mg/liter, mirroring methane production in these systems. After day 70, the decrease in COD concentration in the control stopped, and the level remained fairly constant at approximately 40 mg/liter until the conclusion of the experiment. The soluble COD fraction (⬃40 mg/liter) that remained in the control after day 70 probably represents compounds that were not biodegradable in the sediment slurries within the experimental timeframe. The COD concentration in the 3-CB-amended culture also stopped declining around this time (70 days) and leveled off at approximately 81 mg/liter until day 91. Significant transformation of 3-CB was first detected in the sediment community during this period of relatively stable COD concentration in the amended culture (compare Fig. 1A and D), and the complete removal of 3-CB was reflected by a decrease in COD of approximately 40 mg/liter. A second dose of 3-CB was added to the amended culture on day 107, which was reflected in the high COD measurement on day 114. COD and 3-CB declined in parallel from day 114 to the end of the experiment. COD was not measured in the 3-CB-amended digester culture and associated control. To directly evaluate the potential importance of selective
FIG. 2. Characterization of a digester culture during adaptation to and transformation of 3-chlorobenzoate (3-CB). (A) 3-CB in a single 2-liter culture (filled circles), triplicate 160-ml cultures (open diamonds and circles and crosses), and a single 2-liter sterile control (filled triangles); (B) headspace H2; (C) CH4; (D) total SSU rRNA; and (E) Desulfomonile tiedjei-like SSU rRNA. (B to E) Each datum point represents the concentration in a single 3-CB-amended 2-liter culture (filled circles) or unamended live control (open triangles).
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enrichment in the adaptation process, the abundance of populations involved in biodegradation should be monitored. Mineralization of 3-CB as a sole growth substrate can be carried out by microorganisms mediating reductive dehalogenation of 3-CB, syntrophic benzoate fermentation, and methanogenesis (11). Desulfomonile tiedjei is the only freshwater organism that has been shown to conserve energy from the reductive dehalogenation of 3-CB under anaerobic conditions in pure culture (10). Several pieces of evidence indicate that D. tiedjei was present in the sediment and digester cultures following their adaptation to 3-CB. For example, D. tiedjei-like sequences were detected in denaturing gradient gel electrophoresis fingerprints generated using Bacteria-specific primers and DNA obtained from the 3-CB-degrading cultures reported here (data not shown) as well as from triplicate anaerobic cultures that repeatedly degraded additions of 3-CB with an inoculum from the same site used to establish the sediment cultures described in this study (4). Amplification of DNA using D. tiedjei-specific primers yielded no product for samples obtained from the digester control throughout the course of the experiment, as shown in Fig. 3 for DNA extracted on days 44, 75, 95, and 126. Further, D. tiedjei-like sequences were not detected in the 3-CB-amended digester culture before the onset of 3-CB biodegradation on day 53, as shown in Fig. 3 for DNA extracted on day 44. However, D. tiedjei-like sequences were detected in the 3-CB-degrading culture on days 75, 95, and 126 (Fig. 3) and other sampling dates following the adaptation event (data not shown). The potential importance of Syntrophus populations (whose hallmark is syntrophic benzoate fermentation [21]) in the adapted 3-CB-degrading sediment and digester cultures had previously been recognized through the use of SSU rRNA gene fingerprinting techniques (4). The production of methane in the sediment and digester cultures indicated that methanogenic populations were also active in these systems. Therefore, probes that specifically target the SSU rRNAs of D. tiedjei, members of the genus Syntrophus, and the archaea (presumably represented primarily by methanogens in these cultures) were used to quantify the abundance of these potentially important populations. In addition, a universal probe was used to quantify total SSU rRNA as a general measure of growth activity averaged over the entire sediment or digester community. The results of the hybridizations conducted with the universal probe and rRNA extracted from sediment and digester cultures are shown in Fig. 1E and 2D, respectively. Although the initial levels of total SSU rRNA were orders of magnitude higher in the digester culture and control than in the sediment culture and control, the general trends in the total SSU rRNA data were the same for the sediment and digester communities. During the adaptation period preceding 3-CB biodegradation, a net decrease in the total SSU rRNA levels in all of the cultures and controls was observed, and the total SSU rRNA levels in the controls were similar to the concentrations in the corresponding 3-CB-amended culture. In the amended sediment culture, a significant peak in total SSU rRNA levels coincided with the removal of a second 3-CB addition, suggesting that the community benefited from the net release of electrons derived from 3-CB biodegradation (Fig. 1E). In contrast, total SSU rRNA levels in the sediment control remained
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FIG. 3. Detection of Desulfomonile tiedjei-like SSU rRNA genes in a 3-chlorobenzoate (3-CB)-amended digester culture and live control following PCR amplification and agarose gel electrophoresis. SSU rRNA gene sequences were amplified using D. tiedjei-specific primers (S-S-Dtied-0059-a-S-22 and S-S-Dtied-1032-a-A-22 [13]). Lanes: M, 100-bp DNA molecular size markers (Pharmacia, Piscataway, NJ); 1, live control, day 44; 2, 3-CB-amended culture, day 44; 3, live control, day 75; 4, 3-CB-amended culture, day 75; 5, live control, day 95; 6, 3-CB-amended culture, day 95; 7, live control, day 126; and 8, 3-CBamended culture, day 126. Significant removal of 3-CB was first noted in the amended culture on day 53.
constant at approximately 50 ng/ml, beginning on day 49. The effect of 3-CB biodegradation on total SSU rRNA levels in the 3-CB-amended digester culture was not readily apparent, presumably because the total SSU rRNA levels were much higher in the digester culture and control than in their sediment counterparts. There was no obvious increase in total rRNA levels in the digester culture after 3-CB biodegradation began relative to the levels in the control or in the amended culture during the adaptation period. In general, the abundance of SSU rRNA targeted by probes specific for the three populations believed to be important in anaerobic mineralization of 3-CB followed trends that were similar to those observed in the total SSU rRNA. This was the case for results obtained with the Syntrophus- and Archaeaspecific probes, which have been reported previously (4). In addition, the levels of D. tiedjei in the 3-CB-amended digester culture and control decreased overall during the adaptation period (Fig. 2E). However, during periods of 3-CB biodegradation, the amounts of rRNA targeted by the D. tiedjei-specific probe in the amended digester culture generally increased and typically exceeded the amounts in the control by greater margins than during the adaptation period. The hybridization conducted with the D. tiedjei-specific probe and rRNA extracted from the amended sediment culture and control samples did not show any clear enrichment in the D. tiedjei population in the amended culture before or after adaptation compared with the control (data not shown). Endogenous substrate depletion experiments. The average length of time preceding removal of 3-CB was evaluated in three sets of digester cultures that differed with respect to their previous exposure to 3-CB and availability of endogenous substrates (Fig. 4). Four cultures were unacclimated to 3-CB and were rich in endogenous substrates. 3-CB removal in this set of cultures is also presented in Fig. 2A. One pair of cultures was unacclimated to 3-CB and had been depleted of endogenous substrates before being exposed to 3-CB. Finally, one pair of cultures had both depleted endogenous substrates and adapted to and transformed 3-CB. As expected, removal of 3-CB was first observed in the adapted controls (ⱕ19 days). Adaptation to 3-CB occurred next (ⱕ41 days) in the unacclimated, substrate-depleted cul-
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tures, which required significantly less time than adaptation of the substrate-rich cultures (ⱕ60 days). DISCUSSION Although seldom demonstrated unequivocally, adaptation of a microbial community to an initially persistent compound is frequently attributed to one or more of the following mechanisms (1, 25): (i) mutation or genetic exchanges, (ii) selective enrichment of a minor population capable of degrading the compound of interest before exposure occurred, (iii) enzyme regulation, and (iv) alleviation of conditions that are not amenable to biodegradation, including the presence of a preferential substrate, i.e., diauxy. Conceivably, each of these adaptation mechanisms could be offered as a plausible hypothesis to explain the extended adaptation periods that precede anaerobic biodegradation of 3-CB in this and other studies (18, 20, 25). In addition, the transfer of an inoculum to fresh growth medium often causes a lag phase in the batch growth cycle (1). The weight of evidence suggests that alleviation of unfavorable conditions in the sediment and digester cultures played the most important role in their adaptation to 3-CB. That is, 3-CB biodegradation in the sediment and digester cultures appears to have been stimulated, at least in part, by the depletion of readily degradable substrates (electron donors) that were endogenous to the sediment and digester environments. The negative impact of readily degradable substrates on the adaptation of microbial communities to pollutants is not without precedent. For example, the addition of a low-molecularweight carbohydrate increased the adaptation periods preceding the aerobic removal of p-nitrophenol by pristine aquifer samples (40), and Kuiper and Hanstveit (24) suggested that marine bacteria degrade 4-chlorophenol only after naturally occurring organic constituents of seawater have been depleted. Evidence that endogenous substrates were preferentially utilized relative to 3-CB included increasing CH4 levels in the digester and sediment communities (Fig. 1C and 2C) and decreasing COD concentrations in the sediment community (Fig. 1D) during the adaptation periods. CH4 and COD concentrations leveled off concomitantly with the onset of 3-CB biodegradation, suggesting that the readily degradable fraction of endogenous substrates was largely depleted before 3-CB metabolism was initiated. Adaptation to 3-CB also occurred significantly faster in digester cultures that had previously been depleted of readily degradable substrates compared with digester cultures that were rich in endogenous substrates (Fig. 4). These results are consistent with the idea that depletion of readily degradable substrates stimulated removal of 3-CB. Key populations within the substrate-depleted cultures were presumably negatively impacted by their extended incubation (⬃150 days) without endogenous substrates or 3-CB. This probably explains, at least in part, why 3-CB biodegradation did not begin immediately within the substrate-depleted cultures. To verify this idea, 3-CB biodegradation was also monitored in 3-CB-adapted cultures following approximately 6 weeks of incubation without endogenous substrates or 3-CB (Fig. 4). Clearly, resumption of 3-CB biodegradation in the adapted cultures was also delayed after they were devoid of substrates for a relatively long time. As previously mentioned, other potential explanations for
FIG. 4. 3-Chlorobenzoate (3-CB) biodegradation in substrate-depleted, unacclimated digester cultures (triangles); substrate-rich unacclimated digester cultures (circles); and 3-CB-adapted digester cultures (squares). Each datum point represents the average concentration in two (triangles and squares) or four replicates (circles). Error bars represent the 95% confidence interval. Arrows indicate the first date on which the average 3-CB concentration was at least 5% lower compared with previous measurements.
the observed adaptation events in the 3-CB-degrading cultures can be offered but are less likely than preferential utilization of endogenous substrates. Lag phases resulting from the transfer of a bacterial inoculum to fresh growth medium typically last for several hours at most and, therefore, cannot explain the lengthy adaptation periods preceding the biodegradation of many organic compounds (1). A role for random genetic changes in bringing about 3-CB biodegradation can also be ruled out because adaptation was reproducible among sediment and digester culture replicates (Fig. 1A and 2A). Similarly, Linkfield et al. (25) eliminated genetic change as a potential adaptation mechanism in anaerobic lake sediments that reproducibly adapted to halogenated benzoates. To explicitly evaluate the possibility that selective enrichment of an initially minor population within the sediment or digester cultures resulted in adaptation when the population became large enough to noticeably affect 3-CB levels, we used a combination of SSU rRNA and SSU rRNA gene-based measures to monitor the sizes of populations thought to be important in the biodegradation of 3-CB relative to the total abundance of the other community members. These populations included D. tiedjei, members of the genus Syntrophus, and methanogens. SSU rRNA gene-based evaluations suggested that D. tiedjei, or a close relative of this organism, played a role in 3-CB biodegradation in the adapted digester and sediment cultures (4). D. tiedjei was never detected in the digester control and could be amplified from the 3-CB-amended culture only after the onset of 3-CB biodegradation (Fig. 3). The results of the hybridization conducted with a D. tiedjei-specific probe and rRNA extracted from the digester culture and control are consistent with the DNA data. As shown in Fig. 2E, D. tiedjei appears to have been modestly enriched in the amended digester culture relative to the control after the onset of 3-CB removal. After biodegradation of a second spike of 3-CB began, the level of D. tiedjei-like rRNA increased further com-
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pared to the control. In contrast, D. tiedjei-like rRNA levels in the amended culture and control decreased overall during the adaptation period. Together the results suggest that an increase in the abundance of the presumptive 3-CB-degrading population occurred after, but not before, the adaptation event. Of course, the SSU rRNA and SSU rRNA gene-based analyses targeting D. tiedjei could not evaluate the potential importance of uncharacterized organisms that might have been present in the cultures and possessed unrecognized 3-CB-degrading abilities. As reported earlier, Syntrophus rRNA levels decreased during the adaptation period in the 3-CB-amended sediment and digester cultures and increased only after the onset of 3-CB biodegradation (4). Of the three populations expected to be important in an adapted 3-CB-degrading culture, only methanogens appeared to increase in abundance prior to the onset of 3-CB biodegradation in the amended sediment culture (4). However, the increase in the activity of methanogens during this period appeared to be associated with metabolism of endogenous substrates rather than 3-CB, and, consequently, was also observed in the control. Biomass and readily degradable endogenous substrate levels were much higher in the digester inoculum than in the sediment, based on a comparison of the total SSU rRNA and CH4 levels, respectively, in the two systems (Fig. 1 and 2). Had selective enrichment of the dehalogenating organism or another key population played an important role in the adaptation of the sediment and digester cultures to 3-CB, it follows that higher cell densities and perhaps the greater total substrate availability would probably have enabled this population to grow and noticeably affect 3-CB levels more quickly in the digester cultures than in the sediment cultures. This was not the case. Similarly, it does not appear that any loss of microbial activity that might have occurred in the sediment during storage contributed significantly to the delays preceding 3-CB biodegradation. Adaptation periods of similar durations were observed in experiments conducted with 3-CB and sediment collected at various times in the year and stored for different periods (data not shown). Too little is currently known about utilization of multiple substrates in mixed cultures to speculate about potential regulatory mechanisms that could explain the preferential utilization of endogenous substrates in the digester and sediment cultures prior to the onset of 3-CB biodegradation. Most experimental and recent theoretical evaluations of microbial growth with substrate mixtures are restricted to pure-culture phenomena (8, 12, 30). Further, relatively few studies have addressed the regulation of anaerobic aromatic compound metabolism in pure cultures (9, 19, 32, 33, 37). Repression of 3-CB reductive dehalogenation in D. tiedjei has been reported previously (28). However, the inhibition was caused by alternative electron acceptors (sulfur oxyanions) rather than electron donors. We considered the possibility that H2 levels could play a role in the control of 3-CB metabolism in the sediment and digester cultures. For example, high H2 levels could inhibit 3-CB removal by making fermentation, a critical step in the anaerobic mineralization of 3-CB, thermodynamically unfavorable. Very low H2 levels could also limit 3-CB removal if H2 serves as the ultimate electron donor for reductive dehalogenation. However, H2 levels did not appear to be related to
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adaptation of the digester and sediment cultures to 3-CB. Headspace H2 levels of 10 to 30 ppm were conducive to reductive dehalogenation of 3-CB in the sediment and digester cultures, and the relatively high initial H2 concentrations decreased to these levels at least 45 days before 3-CB biodegradation began (Fig. 1B and 2B). When considering the fate of pollutants in the environment, it is generally expected that simultaneous, rather than sequential, consumption of natural organic substrates and anthropogenic compounds will occur because dissolved organic carbon can sometimes support the growth or maintenance of the pollutant-degrading organisms (12). Our findings demonstrate that exceptions to this generalization exist in dissimilar anaerobic environments. Substrates that were endogenous to sediment or digester sludge were metabolized before significant 3-CB biodegradation began. While involvement of other adaptation mechanisms, such as selective enrichment, cannot be completely ruled out, in part because uncharacterized organisms with unknown physiologies may have played a role in the mineralization of 3-CB in the sediment and digester cultures, it appears more likely that depletion of the endogenous substrates played the dominant role in initiating 3-CB biodegradation. Further, it is clear that the anaerobic microbial communities studied here, once depleted of endogenous substrates, benefited from the investment of electrons in reductive dehalogenation of 3-CB to form benzoate. Benzoate was then fermented to H2 and acetate, which sustained hydrogenotrophic and acetotrophic populations, including methanogens and perhaps dehalogenating organisms (4, 5). ACKNOWLEDGMENTS This research was supported by the United States Environmental Protection Agency Science to Achieve Results (S.T.A.R.) grant R823351. We thank Barbara MacGregor (Department of Marine Sciences, University of North Carolina—Chapel Hill) for help with analysis of the hybridization data. The assistance of the Clavey Road Wastewater Treatment Plant personnel in obtaining samples of the digester contents is also appreciated. REFERENCES 1. Alexander, M. 1999. Biodegradation and bioremediation, 2nd ed. Academic Press, San Diego, Calif. 2. Amann, R. I., L. Krumholz, and D. A. Stahl. 1990. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172:762–770. 3. American Public Health Association, American Water Works Association, and Water Environment Federation. 1998. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, American Water Works Association, and Water Environment Federation, Washington, D.C. 4. Becker, J. G., G. Berardesco, B. E. Rittmann, and D. A. Stahl. 2005. The role of syntrophic associations in sustaining anaerobic mineralization of chlorinated organic compounds. Environ. Health Perspect. 113:310–316. 5. Becker, J. G., G. Berardesco, B. E. Rittmann, and D. A. Stahl. 2001. Successional changes in an evolving anaerobic chlorophenol-degrading community used to infer relationships between population structure and systemlevel processes. Appl. Environ. Microbiol. 67:5705–5714. 6. Becker, J. G., D. A. Stahl, and B. E. Rittmann. 1999. Reductive dehalogenation and conversion of 2-chlorophenol to 3-chlorobenzoate in a methanogenic sediment community: implications for predicting the environmental fate of chlorinated pollutants. Appl. Environ. Microbiol. 65:5169–5172. 7. Boyd, S. A., and D. R. Shelton. 1984. Anaerobic biodegradation of chlorophenols in fresh and acclimated sludge. Appl. Environ. Microbiol. 47:272– 277. 8. Brandt, B. W., F. D. L. Kelpin, I. M. M. van Leeuwen, and S. A. L. M. Kooijman. 2004. Modelling microbial adaptation to changing availability of substrates. Water Res. 38:1003–1013.
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