Apr 4, 1988 - Microbial degradation of halogenated hydrocarbons: a biological solution to the problem? Angew. Chem. Int. Ed. Engl. 25:779-789. 15. Neilson ...
Vol. 54, No. 7
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1988, p. 1864-1867 0099-2240/88/071864-04$02.00/0 Copyright © 1988, American Society for Microbiology
Microbial Degradation of Xenobiotic, Aromatic Pollutants in Humic Water PER LARSSON,* LENNART OKLA, AND LARS TRANVIK
Department of Ecology, Limnology, Box 65, University of Lund, S-221 00 Lund, Sweden Received 19 January 1988/Accepted 4 April 1988
The microbial degradation of a number of "4C-labeled, recalcitrant, aromatic pollutants, including trichloroguaiacol and di-, tri-, and pentachlorophenol, was investigated in aquatic model systems in the laboratory. Natural, mixed cultures of microorganisms in the water from a brown-water lake with a high content of humic compounds mineralized all of the tested substances to a higher degree than did microorganisms in the water from a clear-water lake. Dichlorophenol was the most rapidly degraded pollutant.
There is great concern about the deleterious effects of aromatic, chlorinated hydrocarbons, such as chlorinated phenols, on natural environments. Although acute toxicity is uncommon, these substances cause sublethal damage, e.g., reduced reproduction and physiological disturbances, to a wide range of organisms, thereby reducing the competitive abilities of these organisms (13). Another characteristic feature of the synthetic chlorinated hydrocarbons is their environmental persistence. Microorganisms from polluted environments have been isolated and used to break down chlorinated hydrocarbons (for reviews, see references 3, 14, and 18). Bacterial strains from sediments from the polychlorinated biphenyl-contaminated Hudson river (New York, U.S.A.) were able to degrade a majority of the congeners included in a commercial polychlorinated biphenyl oil (2). Chlorinated phenols and guaiacols can be degraded by mixed bacterial cultures obtained from areas polluted by bleach plant effluents which contain these compounds (15). Mixed bacterial cultures originating from soil contaminated by polychlorinated phenols, which are used as wood preservatives in sawmills, degrade pentachlorophenol (20). Certain microorganisms may increase their fitness in polluted environments by degrading chlorinated, aromatic pollutants. Is it possible, however, that microorganisms in relatively pristine environments can degrade persistent, aromatic pollutants as well? Considering the ubiquity of highly condensed, aromatic compounds, such as lignin residues and humic substances, and, further, assuming a certain degree of generality between these compounds and aromatic pollutants, such a hypothesis seems reasonable. Brown-water lakes, which receive humic substances from forests and bogs, are examples of habitats where natural aromatic compounds (humic substances) are abundant. The humic substances, which give the water its characteristic brown color, are recalcitrant (6), and a major fraction contain aromatic, phenolic structures (10). Nevertheless, microorganisms are able to degrade humic substances in aquatic environments (7, 16), and there is a positive correlation between bacterial biomass and humic substance content in lakes (11; L. Tranvik, Microb. Ecol., in press). Bacteria growing in humic water in mixed batch cultures degrade phenol at higher rates than do bacteria growing in clear water (19). This may reflect higher degradation rates for phenolic structures of the humic compounds as well (19). *
On the basis of the structural similarities between synthetic, chlorinated, aromatic pollutants and natural, aromatic humic substances, we suggest that recalcitrant, aromatic pollutants are degraded by microorganisms in aquatic ecosystems that contain high levels of humic substances. This hypothesis presumes that the microbial enzyme systems active in the degradation of aromatic humic compounds are nonspecific in their attacks on ring structures, resulting in the degradation of aromatic pollutants. Degradation studies were carried out by a technique, similar to that used by Bumpus et al. (5) and Eaton (8), which detects degradation of pollutants by measuring the respiratory release of 14CO2. Briefly, the '4C-labeled, chlorophenolic pollutants were added to 100 pl of ethanol, and the solvent was gently evaporated. A 50- to 200-ml sample of fresh, prescreened (through a 10-,um-pore-size plankton net) humic or clear lake water with its natural content of microorganisms was added to each of several 250-ml flasks with Teflon stoppers (Fig. 1). Air was drawn into the systems, and the water phase was bubbled for 15 min once every 6 h to strip out '4CO2 developed during mineralization of the added "4C-labeled substance. The 14CO2 was trapped in a C02absorbing solution (Carbosorb; Packard Instrument Co., Inc.), and the "4C- radioactivity was measured with a liquid scintillation counter (LS 1801; Beckman Instruments, Inc.). The C02-absorbing solution was changed regularly to obtain the degradation rates for the pollutants. To prevent volatilAir
Filter (XAD-2 + polyurethane)
CO -trap FIG. 1. Experimental setup for the degradation studies. Air was sucked into the system to strip the water phase of 14CO2 generated during mineralization of added 14C-labeled compounds. The 14CO2 was subsequently caught in a C02-absorbing solution. Nondegraded, volatilized 14C-labeled compounds were trapped on an XAD-2/ polyurethane filter.
Corresponding author. 1864
VOL. 54, 1988
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FIG. 2. Degradation of chlorophenolic compounds as cumulative percentages of "'CO2 generated from added "'C-labeled substances over time. HgCl2 was added randomly to some systems after 14 days to inhibit microbial activity; these systems served as abiotic controls. Each point represents a mean value for two (abiotic controls) or three systems. (a) DCP; (b) PCP; (c) TCP; (d) TCG. Symbols: *, systems with humic water; O, systems with clear water; A, systems with humic water plus HgCl2; A, systems with clear water plus HgCl2.
ized, nonmineralized, persisteht pollutants from reaching the carbon dioxide trap, a filter (XAD-2 with 5-mm-thick polyurethane foam plugs at both ends) capable of adsorbing the pollutants was inserted between the model system and the trap. Incubations were carried out at room temperature (ca. 200C). Water was taken from two lakes, which differed greatly in their humic-compound contents (as indicated by their water color [21]), situated in a forest area in southern Sweden: the brown-water lake Frejen (water color, ca. 160 mg of Pt/liter; dissolved organic carbon, ca. 21 mg/liter; pH ca. 5.2) and the clear-water lake Fiolen (water color, ca. 10 mg of Pt/liter; dissolved organic carbon, ca. 7 mg/liter; pH ca. 5.5). No point sources of chlorinated compounds exist in the drainage areas of the lakes, so they presumably receive pollutants only from the atmosphere. Some general characteristics of these lakes are given by Tranvik (in press). Aseptic techniques were not used during sampling and experiments; our approach was, rather, to monitor the degradation of the added substances by any microbial community developing under the conditions prevailing in the waters. The experiments started within 24 h after sampling.
The following four substances of different degrees of recalcitrance and lipophilicity were studied: (i) 3,4,5-trichloroguaiacol (TCG), which had a 14C-labeled methyl group (specific activity, 2.4 Ci/mol); (ii) 3,4-dichlorophenol (DCP; specific activity, 26.9 Ci/mol); (iii) 2,4,5-trichlorophenol (TCP; specific activity, 9.0 Ci/mol); and (iv) pentachlorophenol (PCP; specific activity, 13.5 Ci/mol). All chlorophenols were uniformly 14C labeled in the ring. From 1 to 2 pRCi of the substances was added to each system. The resulting concentrations of the phenolic pollutants were 30 to 537 ,ug/liter. Microbial mineralization of each substance was monitored in five model systems each for both of the water types. After 14 days, HgCl2 (1 ml of a 5% solution) was added to two model systems in each group of five to inhibit microbial activity and, thereby, mineralization of the xenobiotic pollutants. The systems to be poisoned with HgCl2 were chosen randomly before the start of the experiment. All substances studied were more readily mineralized in humic lake water than in water from the clear lake. Mineralization rates for the chlorophenols were significantly higher in systems containing humic water than in corresponding clear-water systems (Wilcoxon matched-pairs
APPL. ENVIRON. MICROBIOL.
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FIG. 3. Degradation rates of chlorophenolic compounds. Error bars indicate standard deviations for triplicate systems. (a) DCP; (b) PCP; (c) TCP. Symbols: *, systems with humic water; OI, systems with clear water.
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signed-rank test, P = 0.004). DCP was the most labile of the chlorinated phenols; the resistance to microbial mineralization increased in the order DCP < PCP < TCP < TCG. In the humic lake water, 15% of the added DCP was respired within 119 days (Fig. 2a), compared with 7% in the clear lake water. In the abiotic controls, an average of 0.7% of the DCP was recovered in the CO2 traps. The proportion of total PCP respired was 10% in the humic lake water, 5% in the clear lake water, and less than 2% in the abiotic controls (Fig. 2b). For TCP, the corresponding percentages were 6% in the systems containing humic lake water, 3% in those containing clear lake water, and less than 2% in the abiotic controls (Fig. 2c). The mineralization of TCG ceased after 53 days (Fig. 2d). By that time, about 6% of the added substance had been converted to CO2 in the humic lake water. Corresponding estimates for clear-water and HgCl2-poisoned systems are 2% and 1%, respectively. When HgCl2 was added to the systems, the microbial degradation of the polychlorinated phenols ceased. Before addition of the HgCl2, 62 to 69% of the total degradation recorded during the experiment had occurred. After the systems were poisoned with HgCl2, the degradation measured was probably attributable to chemical decomposition. Because the chlorinated phenols are ionized within the pH intervals prevailing in the model systems, volatilization of the compound is not likely to occur. HgCI2 was added to some TCG-containing systems at the start of the experiment. In these abiotic control systems, small amounts of radioactivity left the water phase. The highest rate of mineralization was recorded for DCP in the humic-compound-containing systems; 90,200 dpm of the compound was converted to '4CO2 per day (Fig. 3a), corresponding to a respiration rate of 246 ng of DCP per day. In humic water, the microbial mineralization continuously increased from the start of the experiment until day 42 and remained high until day 98. In the clear-water systems, 48 to 140 ng was respired per day. The rate was initially high, but it gradually decreased during the rest of the experiment (Fig.
3a). In the humic-compound-containing systems, the rate of PCP mineralization reached 32 ng/day by day 42 (Fig. 3b), after which it remained rather constant at 18 to 36 ng/day. In the clear-water systems, the rates were low throughout the experiment (6 to 18 ng/day). The rates of TCP mineralization in humic water increased to 31 ng/day by day 42 and then continuously decreased (Fig. 3c). Rates were consistently lower in the clear-water systems, i.e., 4 to 9 ng/day. After day 92, mineralization of all chlorophenols decreased. The rates of TCG mineralization were between 47 and 230 ng/ day. Contrary to the rates of mineralization of other chlorophenolic compounds, the rate was high in the initial phase of the incubation and then decreased in the latter part (until day 53). The observed changes in mineralization rates over time may reflect the effects of enclosing the water. Within a day after confinement, planktonic bacteria show altered structure and activity (9). Because the systems initially included all microbial components smaller than 10 ,um in diameter that were present in the lakes, there was probably a continuous cycling of organic carbon through osmotrophs and phagotrophs, as well as a simultaneous slow decline in the amount of organic carbon, owing to mineralization. Assuming that the microorganisms preferentially degraded the most labile components of the available organic carbon, there should have been a successive degradation of more-refractive compounds with time as the less persistent compounds were gradually mineralized. Such a pattern has been shown in mixed batch cultures with natural bacterioplankton from a humic lake, in which glucose degradation was highest during exponential growth, this was followed by a peak in the degradation of a more-refractive compound, phenol, when the bacterial growth rate declined owing to decreased substrate availability (19). This pattern would correspond to that observed for the chlorophenols studied here; i.e., the degradation rates for aromatic pollutants increased with time, presumably in response to a decrease in the availability of more easily degradable substrates. Interestingly, TCG, which, unlike the other tested compounds, was not ringlabeled but was labeled in its methyl group, was respired at a comparatively higher rate during the early phase of incubation. A difference in the mechanisms by which the methyl substituent and the aromatic nucleus are degraded could be responsible for the initially higher respiration rate in the TCG-containing systems. These experiments demonstrated that aromatic, chlorinated compounds are degraded to CO2 to a greater extent in
VOL. 54, 1988
the water from a humic lake than they are in the water from a clear-water lake. When the water was poisoned with HgCl2, degradation declined sharply, indicating that it is mainly a biological process. Thus, the microbial mineralization of these compounds was higher in humic water than in clear water. The relatively higher rate of microbial mineralization of aromatic compounds in the humic water may be due to the selection for microorganisms capable of degrading aromatic compounds in a natural environment rich in such compounds. Similarly, Bumpus et al. (5) and Eaton (8) proposed that the ligninolytic enzyme system of the white rot fungus, Phanerochaete chrysosporium, was responsible for the degradation of several persistent aromatic pollutants as well as lignin. Tranvik and Hofle (19) suggested that bacterial assemblages in water from a humic lake degraded phenol to a greater extent than did assemblages in water from a clear-water lake owing to the relatively better ability of the former to degrade phenolic structures associated with naturally occurring humic substances. Thus, the presence of naturally occurring microbial substrates with structural similarities to polluting compounds might have an influence on the fate of pollutants in the environment. This hypothesis is supported by the finding that adaptation of microbiota to a certain substance may increase the ability of the cells to degrade a structurally related compound as well. Thus, Brunner et al. (4) recorded increased biodegradation of polychlorinated biphenyls in soil enriched with nonchlorinated biphenyls, and Shimp and Pfaender (17) demonstrated increased degradation of several substituted phenols by microbial communities adapted to nonsubstituted phenol. Here, we applied this "analog enrichment concept" to indigenous compounds of natural ecosystems versus anthro-
pogenic, synthetic compounds. No effort was made in this study to assess either the structural or growth characteristics of the microbiota. Although bacteria are generally regarded to be the dominant microheterotrophs responsible for the degradation of dissolved organic carbon in pelagic waters (1), we cannot exclude the possibility that pollutant-degrading fungi developed in the experimental systems. In addition, it was not possible to determine whether the respiration of the radiolabeled chlorinated substances was due to cometabolism (12), with less-refractive compounds serving as carbon sources, or whether the radiolabeled compounds per se constituted carbon sources for the microbiota. The microbial species active in the degradation of pollutants in natural humic environments are probably not the same as those enriched from contaminated sites. Thus, the potential for natural microbial communities of essentially unpolluted humic lakes to degrade aromatic, chlorinated hydrocarbons should be of interest in more-applied aspects of the degradation of xenobiotic pollutants. We thank G. Bengtsson, H. Blanck, J. F. McCarthy, and A. Sodergren for constructive criticism of the earlier drafts of this manuscript. D. Tilles greatly improved the English.
This work was financed by the National Swedish Environmental Protection Board.
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