National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan1
Author for correspondence: Yuichi Suwa. Tel: +81 298 61 8747. Fax: +81 298 61 8309. e-mail: y-suwa{at}aist.go.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: anaerobic degradation, chlorophenols, denitrification, enrichment culture
Abbreviations: CP, chlorophenol; DCP, dichlorophenol
a Present address: Biosystem Engineering Laboratory, Department of Chemical Engineering, Korean Advanced Institute for Science and Technology, 373-1 Kusong-Dong, Yusung-Gu, Taejon 305-701, Korea.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chlorophenols (CPs) are common environmental pollutants arising from the extensive use of wood preservatives, herbicides and fungicides (Valo et al., 1985 ), and are also found in pulp bleaching effluents and industrial wastewater (Kringstad & Lindström, 1984
; Valo et al., 1984
). Anaerobic degradation of CPs has mainly been studied under methanogenic conditions in anaerobic sediments (Zhang & Wiegel, 1990
) with microbial consortia obtained from various environments (Boyd & Shelton, 1984
; Gibson & Suflita, 1986
; Mikesell & Boyd, 1986
; Genthner et al., 1989a
; Madsen & Aamand, 1992
; Mohn & Kennedy, 1992
; Nicholson et al., 1992
; Takeuchi et al., 2000
) and anaerobic reactor systems (Woods et al., 1989
; Juteau et al., 1995
). These studies have indicated that CPs are initially dechlorinated to less chlorinated phenols via a reductive dechlorination, and, then, ultimately mineralization to methane and CO2 in some cases. Recently, some anaerobic bacteria capable of aryl-dechlorination have been obtained from methanogenic enrichments (Madsen & Licht, 1992
; Cole et al., 1994
; Utkin et al., 1995
; Sanford et al., 1996
). A few studies have been conducted to investigate the anaerobic degradation of chloroaromatics under reducing conditions other than methanogenic conditions such as denitrifying conditions, sulfate-reducing conditions and iron reducing conditions (Genthner et al., 1989b
; Häggblom & Young, 1990
, 1995
; Häggblom et al., 1993
; Kazumi et al., 1995
). These studies established sulfate- and iron-reducing enrichments capable of degrading CPs, and demonstrated that the anaerobic degradation of CPs was coupled with sulfate reduction (Genthner et al., 1989b
; Häggblom & Young, 1990
, 1995
; Häggblom et al., 1993
) or iron reduction (Kazumi et al., 1995
). Anaerobic degradation of 2-CP was observed in a sediment or a water sample incubated with nitrate (Genthner et al., 1989b
; Häggblom et al., 1993
), but the capability could not be sustained by re-feeding with 2-CP or by transferring the sample culture to fresh medium. Thus, there has been no substantial evidence for anaerobic degradation of CPs coupled with denitrification; thereby CPs have been regarded as recalcitrant under denitrifying conditions until now. Obtaining evidence of CP degradation under denitrifying conditions was the aim of this study. First, we attempted to establish enrichment cultures with sustained capability to degrade CPs under denitrifying conditions. Second, the kinetics and physiological characteristics of the enrichment cultures were quantitatively evaluated with respect to CP degradation under denitrifying conditions.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Media.
The culture medium was prepared by strictly anaerobic techniques and used throughout the study. The MSM used in this study was composed of 2 g Na2HPO4, 1·0 g KH2PO4, 0·5 g NH4Cl and 00·43 g NaNO3 in 1 l distilled water. After autoclaving, the medium in the reservoir was quickly chilled with running water under flushing N2 that is passed through an oxygen-trap filter (Chemical Research Supplies), and then 1 ml EDTA-chelated trace element mixture, 1 ml alkaline-earth-metal solution (100 g MgCl2.6H2O, 25 g CaCl2.2H2O in 1 l distilled water), 1 ml Na2SeO3/Na2WO4 solution (Widdel & Bak, 1992 ), vitamin solutions (Widdel & Bak, 1992
) and 5 ml bicarbonate solution (84 g NaHCO3 in 1 l distilled water) were added to 1 l MSM in an anaerobic chamber. The trace element solution was composed of 5·2 g EDTA, 0·45 g FeCl2.4H2O, 52 mg ZnCl2, 0·19 g CoCl2.6H2O, 0·1 g MnSO4.5H2O, 24 mg NiCl2.6H2O, 29 mg CuCl2.2H2O, 36 mg Na2MoO4.2H2O and 30 mg H3BO3 in 1 l distilled water. As a reducing agent, Na2S.9H2O was added to the medium as a stock solution (0·2 M) to a final concentration of 0·2 mM. The pH of the media was adjusted to 7·2. The stock solutions (0·5 M) of CPs were prepared by dissolving them in 0·2 M NaOH.
Enrichment culture.
An aliquot of the sludge biomass (20 mg dry wt), pre-treated as mentioned above, was transferred to a 68 ml serum bottle containing 50 ml MSM with 5 mM nitrate in an anaerobic chamber. The bottle was tightly sealed with a butyl rubber stopper and an aluminium seal before exposure to ambient air. Thus, the resultant headspace of the bottle was nominally the same as that of the anaerobic chamber. A filter-sterilized stock solution of either 2-CP, 3-CP, 4-CP, 2,4-dichlorophenol (DCP) or 2,6-DCP was injected using a syringe through the butyl rubber stopper to provide a final concentration of 0·1 mM in the anaerobic chamber. To avoid oxygen contamination during the withdrawal of culture samples, the headspaces of the bottles were kept under positive pressure by an additional supply of oxygen-free nitrogen up to 2030 KPa. All cultures were incubated in the dark at 25 °C. When all the CP supplied to the bottle was degraded, 10 ml of the CP-degrading culture in the bottle was transferred into 68 ml bottles containing 40 ml freshly prepared MSM with 5 mM nitrate. The same CP species was added to bottles inoculated with a CP-degrading culture to a final concentration of 0·1 mM, and no CP was added to the other bottles to serve as a control.
Degradation of 2-CP under other anaerobic conditions.
Biomass from the enrichment culture that degraded 2-CP in the once-transferred cultures was collected by centrifugation (7000 g for 10 min) from a bottle containing 50 ml culture broth and washed three times with nitrate-free MSM to remove nitrate. The washed biomass pellet was placed in the anaerobic chamber and suspended in nitrate-free MSM. Fifty millilitres of the biomass suspension was transferred to each of six 68 ml serum bottles and 2-CP added to a final concentration of 70 µM. Of the six bottles, two were anaerobically spiked with nitrate (5 mM), two with sulfate (5 mM) and the remaining two with neither nitrate nor sulfate.
Analytical procedures.
During incubation, 1 ml of each culture fluid was anaerobically collected several times using a syringe injected through a butyl rubber stopper. Sampling was carried out in the anaerobic chamber. Samples were filtered through a PVDF 0·22 µm pore-size Millipore filter and stored at -20 °C until use. CPs were analysed using a high-performance liquid chromatograph (Hewlett Packard) equipped with a C-18 column and a diode-array detector for monitoring substances at 280 nm. A mixture of water/methanol/acetic acid (40:60:1, by vol.) was used as a running buffer for HPLC analysis, the flow rate of which was 1 ml min-1. Nitrate and nitrite were analysed using an ion chromatograph (IC7000, Yokogawa) equipped with a conductivity detector and an anion exchange column, with 3 mM sodium bicarbonate buffer at a flow rate of 1 ml min-1. Nitrous oxide was quantitatively sampled using a gas-tight syringe and analysed using a gas chromatograph (HP5890, Hewlett Packard) equipped with ECD and 3 m stainless-steel column (3·2 mm external diameter) packed with Porapak Q50/80. A gas mixture comprising 95% argon and 5% methane was used as the carrier gas, the flow rate of which was 30 ml min-1.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
During the degradation of 2-CP in original and transferred cultures, no UV-absorbable intermediates such as phenol or benzoate were detected by HPLC analysis. This suggests that 2-CP degradation in our enrichment cultures is distinguished from transformation of CPs to less- or non-chlorinated phenol via a reductive dechlorination usually observed under methanogenic conditions. To our knowledge, it has rarely been observed that a microbial enrichment culture capable of degrading CP is sustained under denitrifying conditions, which has limited the microbiological study of this subject. Thus, these 2-CP-degrading cultures obtained here may be good materials for further work on this subject.
Stoichiometry of 2-CP degradation and nitrate reduction
Fig. 1(a) shows that the 2-CP-degrading culture originally inoculated with sludge C, which was transferred twice to a freshly prepared medium (second subculture), degraded 0·1 mM 2-CP in 168 d. As shown in Fig. 1(b)
, nitrate was consumed concomitantly during 2-CP degradation, and nitrite transiently accumulated. Another intermediate in the denitrification pathway, nitrous oxide, was also detected in test cultures to which 2-CP was added (Table 2
). It is notable that the calculated number of electrons consumed via denitrification appeared to be stoichiometric to 2-CP degradation all the way through the experiment (Fig. 1c
). Nitrate cannot be assimilated as it is but should be reduced to ammonia before assimilation. Thus, the growing population of bacterial cells in a 2-CP-degrading culture would preferentially assimilate the ammonia (10 mM) added to the media. Rice & Tiedje (1989)
demonstrated that ammonium salts contained in the medium at a high concentration inhibited assimilatory nitrate reduction. Therefore it is more likely that nitrate would be consumed during denitrification than during assimilation. Even though nitrate was used for assimilation of cellular materials, its level is much lower than that used as an electron acceptor in the degradation of 2-CP. Assuming all of the 2-CP (5 µmol) and nitrate could each be solely assimilated into the cell biomass composed of C5H7O2N (Hoover & Porges, 1952
), 6 µmol nitrate could be consumed. The actual observed amount of nitrate consumed by each 2-CP-degrading culture (Table 2
) was much larger than this value. Thus, the amount of nitrate consumed was not explained in terms of assimilation, but in terms of another mechanism, i.e. denitrification.
|
|
Regarding results obtained for a 2-CP-degrading denitrifying culture originally inoculated with sludge C, it was estimated that a mean of 143·8 µmol electrons was consumed by denitrification (Table 2). The theoretical amount of electrons produced via 2-CP oxidation was also calculated for each 2-CP degrading culture under denitrifying conditions (Table 2
). In theory, complete oxidation of 1 µmol 2-CP would provide 26 µmol electrons. For the 2-CP-degrading culture originally inoculated with sludge C, 127·4 µmol electrons would theoretically be produced from 4·9 µmol 2-CP consumed, which is similar to the estimated amount of electrons consumed (143·8 µmol). As shown in Table 2
, the amount of electrons consumed via dissimilatory nitrate reduction was similar to that theoretically produced by 2-CP degradation for other cultures, suggesting that nitrate reduction was apparently accompanied by 2-CP degradation. Other previous studies showed anaerobic degradation of 2-CP in denitrifying enrichment cultures (Genthner et al., 1989b
; Häggblom et al., 1993
)
, however, these studies did not demonstrate the consumption of nitrate during degradation of 2-CP, which may suggest that the substrate used was coupled to denitrification. The present study would provide information on this aspect.
Apparent specific growth rate of micro-organisms responsible for 2-CP degradation
The shape of the 2-CP degradation curve shown in Fig. 1(a) indicates that the 2-CP degradation rate increased with incubation time, possibly as a result of growth of micro-organisms responsible for 2-CP degradation and/or release from substrate inhibition. The experimentally obtained time course data for 2-CP degradation by the enrichment culture originally inoculated with sludge C could be fitted with equation 1, which assumes exponential growth of 2-CP-degrading microbial population:
![]() | (1) |
where So is the initial substrate concentration, S is the substrate concentration at the incubation time period t, k is the initial 2-CP degradation rate at time 0 and µ is the specific growth rate of micro-organisms responsible for 2-CP degradation. It is notable that the apparent specific growth rate of the 2-CP-degrading microbial population was extremely slow, 0·0139 d-1, representing an apparent doubling time of 50·2 d for the population. This result indicates that the 2-CP degrading microbial population has substantially multiplied no more than three times by incubating the enrichment culture for 150 d, which increased the biomass of micro-organisms responsible for 2-CP degradation by only eight times. This may explain why the incubation period required for degrading 0·1 mM 2-CP was not reduced when 20% of the culture was transferred to a freshly prepared medium (Table 1). Thus, a longer incubation of subcultures would be required to obtain an enrichment culture adequate for isolating the putative 2-CP-degrading organisms, and further microbiological studies on this subject would be difficult because of such a slow rate of 2-CP degradation.
Degradation of 2-CP under other anaerobic conditions
To examine whether 2-CP utilizing enrichment cultures degrade 2-CP under other anaerobic conditions, four first subcultures originally inoculated with sludges B, C, D and E were incubated in an anaerobically prepared medium with nitrate (5 mM) or sulfate (5 mM), or without nitrate or sulfate. The culture originally inoculated with sludge E demonstrated that 2-CP was degraded in the presence of nitrate in 147 d, but was not degraded under other conditions (Fig. 2a). This indicates that the enrichment culture required nitrate for anaerobic degradation of 2-CP. Based on this result and the stoichiometry of 2-CP degradation and nitrate consumption (Table 2
), it is likely that the anaerobic degradation of 2-CP in this enrichment culture was strictly dependent on denitrification.
|
The culture originally inoculated with sludge D degraded 2-CP in the absence of nitrate or sulfate and in the presence of nitrate (Fig. 2b). However, this culture did not degrade 2-CP in the presence of sulfate (Fig. 2b
). When 62 µM 2-CP was degraded in the absence of an electron acceptor, 59 µM phenol was detected, indicating that 2-CP was reductively dechlorinated. One possible explanation for the observation is that this enrichment culture may have retained two pathways for anaerobic degradation of 2-CP: one dependent on denitrification and the other independent of it. Further examination would be necessary to clarify whether the reductive dechlorination, which is basically a denitrification-independent pathway, occurs in the presence of nitrate. However, since phenol was not detected in the culture supplied with nitrate, it is not really evident whether the enrichment culture in the bottle concomitantly underwent two reactions, reductive dechlorination and denitrifying degradation of 2-CP.
Cultures originally inoculated with sludges B and C did not degrade 2-CP in the presence of nitrate or sulfate or in the absence of exogenous electron acceptors (data not shown), although these cultures successfully degraded 2-CP when they were transferred into a fresh medium with nitrate without washing as described in Table 1. The reason why 2-CP was not degraded even in the nitrate-added medium is unclear, but one possible explanation may be the inadequate preparation of the inocula. The sludge samples used as inocula were aerobically washed three times with a nitrate-free medium. This procedure may have unintentionally damaged the degradation capability of the micro-organisms or discharged factors required for degradation.
Conclusion
We have demonstrated that denitrifying conditions supported an anaerobic degradation of CPs in enrichment cultures derived from activated sludge samples. Concomitant consumption of nitrate was observed during 2-CP degradation, the amount of which was stoichiometric, assuming that 1) 2-CP was completely oxidized to CO2 and 2) nitrate was solely consumed as an electron acceptor in denitrification (Fig. 1 and Table 2
). This would be evidence demonstrating that 2-CP degradation is coupled to respiratory denitrification. The strict requirement of nitrate for degradation of 2-CP was observed in a 2-CP-degrading enrichment culture under denitrifying conditions (Fig. 2a
), which provides support for the involvement of the denitrification process in anaerobic degradation of 2-CP. Although the 2-CP-degrading enrichment cultures under denitrifying conditions were subcultured repeatedly, the degradation rate could not significantly be increased, possibly because the apparent growth rate of the 2-CP-degrading microbial population was extremely slow, the apparent doubling time of which was 50·2 d. The degradation capability of the culture once enriched was easily lost, particularly when the biomass of the enrichment culture was washed under aerobic conditions. Thus, we do not yet know the conditions for properly and stably maintaining a 2-CP-degrading enrichment culture under denitrifying conditions. Studies are still needed to determine the contribution of the denitrification process in the anaerobic degradation of CPs in contaminated environments, however, the extremely slow degradation rate may imply that this process could easily be predominated by other anaerobic processes.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Braun, K. & Gibson, D. T. (1984). Anaerobic degradation of 2-aminobenzoate (anthranilic acid) by denitrifying bacteria. Appl Environ Microbiol 48, 102-107.[Medline]
Cole, J. R., Cascarelli, A. L., Mohn, W. W. & Tiedje, J. M. (1994). Isolation and characterization of a novel bacterium growing via reductive dehalogenation of 2-chlorophenol. Appl Environ Microbiol 60, 3536-3542.[Abstract]
Dolfing, J., Zeyer, J., Binder-Eicher, P. & Schwarzenbach, R. P. (1990). Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen. Arch Microbiol 154, 336-341.[Medline]
Frazer, A. C., Coschigano, P. W. & Young, L. Y. (1995). Toluene metabolism under anaerobic conditions: a review. Anaerobe 1, 293-303.
Fries, M. R., Zhou, J., Chee-Sanford, J. & Tiedje, J. M. (1994). Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats. Appl Environ Microbiol 60, 2802-2810.[Abstract]
Genthner, B. R. S., Price, W. A.II & Prichard, P. H. (1989a). Characterization of anaerobic dechlorinating consortia derived from aquatic sediments. Appl Environ Microbiol 55, 1472-1476.
Genthner, B. R. S., Price, W. A.II & Pritchard, P. H. (1989b). Anaerobic degradation of chloroaromatic compounds in aquatic sediments under a variety of enrichment conditions. Appl Environ Microbiol 55, 1466-1471.
Gibson, S. A. & Suflita, J. M. (1986). Extrapolation of biodegradation results to groundwater aquifers: reductive dehalogenation of aromatic compounds. Appl Environ Microbiol 52, 681-688.[Medline]
Häggblom, M. M. & Young, L. Y. (1990). Chlorophenol degradation coupled to sulfate reduction. Appl Environ Microbiol 56, 3255-3260.[Medline]
Häggblom, M. M. & Young, L. Y. (1995). Anaerobic degradation of halogenated phenols by sulfate-reducing consortia. Appl Environ Microbiol 61, 1546-1550.[Abstract]
Häggblom, M. M. & Young, L. Y. (1999). Anaerobic degradation of 3-halobenzoates by a denitrifying bacterium. Arch Microbiol 171, 230-236.[Medline]
Häggblom, M. M., Rivera, M. D. & Young, L. Y. (1993). Influence of alternative electron acceptors on anaerobic biodegradability of chlorinated phenols and benzoic acids. Appl Environ Microbiol 59, 1162-1167.[Abstract]
Heider, J. & Fuchs, G. (1997). Anaerobic metabolism of aromatic compounds. Eur J Biochem 243, 577-596.[Abstract]
Hoover, S. R. & Porges, N. (1952). Assimilation of dairy wastes by activated sludge. II. The equation of synthesis and rate of oxygen utilization. Sewage Ind Wastes 24, 306-312.
Juteau, P., Beaudet, R., McSween, G., Lépine, F., Milot, S. & Bisaillon, J.-G. (1995). Anaerobic biodegradation of pentachlorophenol by a methanogenic consortium. Appl Microbiol Biotechnol 44, 218-224.
Kazumi, J., Häggblom, M. M. & Young, L. Y. (1995). Degradation of monochlorinated and nonchlorinated aromatic compounds under iron-reducing conditions. Appl Environ Microbiol 61, 4069-4073.[Abstract]
Kringstad, K. P. & Lindström, K. (1984). Spent liquors from pulp bleaching. Environ Sci Technol 18, 236A-248A.
Madsen, T. & Aamand, J. (1992). Anaerobic transformation and toxicity of trichlorophenols in a stable enrichment culture. Appl Environ Microbiol 58, 557-561.[Abstract]
Madsen, T. & Licht, D. (1992). Isolation and characterization of an anaerobic chlorophenol-transforming bacterium. Appl Environ Microbiol 58, 2874-2878.[Abstract]
Mikesell, M. D. & Boyd, S. A. (1986). Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms. Appl Environ Microbiol 52, 861-865.[Medline]
Mohn, W. W. & Kennedy, K. J. (1992). Limited degradation of chlorophenols by anaerobic sludge granules. Appl Environ Microbiol 58, 2131-2136.[Abstract]
Nicholson, D. K., Woods, S. L., Istok, J. D. & Peek, D. C. (1992). Reductive dechlorination of chlorophenols by a pentachlorophenol-acclimated methanogenic consortium. Appl Environ Microbiol 58, 2280-2286.[Abstract]
Nozawa, T. & Maruyama, Y. (1988). Anaerobic metabolism of phthalate and other aromatic compounds by a denitrifying bacterium. J Bacteriol 170, 5778-5784.[Medline]
Rabus, R. & Widdel, F. (1995). Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria. Arch Microbiol 163, 96-103.[Medline]
Rice, C. W. & Tiedje, J. M. (1989). Regulation of nitrate assimilation by ammonium in soils and in isolated soil microorganisms. Soil Biol Biochem 21, 597-602.
Rockne, K. J., Chee-Sanford, J., Sanford, R. A., Hedlund, B. P., Staley, J. T. & Strand, S. E. (2000). Anaerobic naphthalene degradation by microbial pure cultures under nitrate-reducing conditions. Appl Environ Microbiol 66, 1595-1601.
Sanford, R. A. & Tiedje, J. M. (1997). Chlorophenol dechlorination and subsequent degradation in denitrifying microcosms fed low concentrations of nitrate. Biodegradation 7, 425-434.
Sanford, R. A., Cole, J. R., Löffler, F. E. & Tiedje, J. M. (1996). Characterization of Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chloro-4-hydroxybenzoate. Appl Environ Microbiol 62, 3800-3808.
van Schie, P. M. & Young, L. Y. (1998). Isolation and characterization of phenol-degrading denitrifying bacteria. Appl Environ Microbiol 64, 2432-2438.
Takeuchi, R., Suwa, Y., Yamagishi, T. & Yonezawa, Y. (2000). Anaerobic transformation of chlorophenols in methanogenic sludge unexposed to chlorophenols. Chemosphere 41, 1457-1462.[Medline]
Utkin, I., Dalton, D. D. & Wiegel, J. (1995). Specificity of reductive dehalogenation of substituted ortho-chlorophenols by Desulfitobacterium dehalogenans JW/IU-DC1. Appl Environ Microbiol 61, 346-351.[Abstract]
Valo, R., Kitunen, V., Salkinoja-Salonen, M. & Räisänen, S. (1984). Chlorinated phenols as contaminants of soil and water in the vicinity of two Finnish sawmills. Chemosphere 13, 835-844.
Valo, R., Kitunen, V. & Salkinoja-Salonen, M. S. (1985). Chlorinated phenols and their derivatives in soil and ground water around wood-preserving facilities in Finland. Wat Sci Tech 17, 1381-1384.
Widdel, F. & Bak, F. (1992). Gram-negative mesophilic sulfate-reducing bacteria,. In The Prokaryotes, 2nd edn, pp. 33523378. Edited by A. Balows and others. New York: Springer.
Woods, S. L., Ferguson, J. F. & Benjamin, M. M. (1989). Characterization of chlorophenol and chloromethoxybenzene biodegradation during anaerobic treatment. Environ Sci Technol 23, 62-68.
Zhang, X. & Wiegel, J. (1990). Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments. Appl Environ Microbiol 56, 1119-1127.[Medline]
Received 21 May 2001;
revised 27 July 2001;
accepted 29 August 2001.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |