- Email: [email protected]
Stimulation of reductive dechlorination for in situ bioremediation of a soil contaminated with chlorinated ethenes
~
Pergamon
PI!: S0273-1223(98)00240-6
War. Sci. Tech. Vol. 37. No.8, pp. 105-110. 1998. © 1998 IA wQ. Published by Elsevier Science Ltd Printed in Great Britain. 0273-1223/98 $19'00 + 0'00
STIMULATION OF REDUCTIVE DECHLORINATION FOR IN SITU BIOREMEDIATION OF A SOIL CONTAMINATED WITH CHLORINATED ETHENES Peter J. M. Middeldorp*, Martine A. van Aalst**, Huub H. M. Rijnaarts*, Fons J. M. Stams*, Han F. de Kreuk***, Gosse Schraa* and TomN. P. Bosma** * Depanment of Microbiology,
Wageningen Agricultural University,
6703 CT Wageningen, The Netherlands
** TNO Institute for Environmental Sciences, Energy Research and Process Innovation, P.O. Box 342, 7300 AH Apeldoorn, The Netherlands *** BioSoil R&D B. V., 3341 U Hendrik Ido Ambacht, The Netherlands
ABSTRACT A soil from a fonner chemical redistribution company, contaminated with mainly chlorinated aliphatics, was studied for bioremediation purposes. Groundwater analyses revealed that the original pollutants, i.e. tetrachloroethene (PeE) and trichloroethene (TCE), were present at levels ranging from 2.3 to 122 mglL. Dichloroethene (DCE), vinylchloride (VC), ethene and ethane were also detected at significant concentrations although they had never been introduced to the soil. Relatively high concentrations of cisDCE as compared to trans-DCE and I,I-DCE indicated that a slow in situ biodegradation had taken place by reductive dechlorination. Laboratory experiments with flow-through soil columns were perfonned to detennine the optimal conditions for the enhancement of reductive dechlorination by the indigenous dechlorinating population. The addition of single electron donors to artificial groundwater resulted in the dechlorination of PCE to TCE and cis-DCE, whereas complete dechlorination to ethene was solely achieved with compost extract added to native groundwater. © 1998 IAWQ. Published by Elsevier Science Ltd
KEYWORDS Perchloroethene; trichloroethene; bioremediation; chlorinated solvents; BTEX; electron donor; compost. INTRODUCTION An important environmental problem is caused by the pollution of soils with PCE and TCE due to their wide spread use in dry cleaning and treatment of metal surfaces soil and groundwater pollution. The ex situ cleanup of these contaminated sites is costly and low-cost remediation strategies - e.g. involving biological methods - are urgently needed. 105
106
P. J. J. MIDDELDORP el al.
Anaerobic reductive dechlorination is proven to be an effective means for the detoxification of both aromatic (Bosma et at., 1988; Dolfing and Tiedje, 1986; Horowitz et al., 1982) and aliphatic (Bosma et at., 1994; Bosma et al., 1996; Middeldorp et at., 1996) halogenated compounds. Laboratory studies indicate that reductive dechlorination of PCE to less chlorinated compounds followed by aerobic treatment is a possible strategy for the clean-up of such sites (Fathepure and Vogel, 1991). However, the complete dechlorination of PCE and TCE via reductive dechlorination is also possible (De Bruin et at., 1992; Distefano et at., 1991) and may be simpler from the engineering point of view. However, the occurrence of the proven carcinogen VC (Verschueren, 1983) as an intermediate and possible end product of dechlorination needs attention. Earlier studies have showed the necessity of the presence of an electron donor to obtain the full dehalogenation of PCE under anaerobic conditions (De Bruin et at., 1992; Mohn and Tiedje, 1990; Mohn and Tiedje, 1992). The current study has been carried out at a heavily polluted site in the Netherlands where houses are going to be built. The aim of the present study was to find the most adequate electron donor for a complete reductive dechlorination of PCE - i.e. to ethene and/or ethane - under the conditions prevailing at the contaminated site. SITE DESCRIPTION The current study has been carried out at a heavily polluted site in the Netherlands where houses are going to be built. The site has been used by a firm redistributing a wide range of products from cleaning agents for metal finishing and for boiler maintenance to products for carbon removal from combustion engines. In addition metal surfaces were treated at the factory as well. These activities caused a soil pollution problem with a wide range of contaminants among which chlorinated solvents including PCE and TCE are the most important. Further contaminants are phenols, dichlorobenzenes, several aromatics such as benzene, toluene and xylenes (Table I) and non-chlorinated solvents with a f1ashpoint above 65°C. The top soil is contaminated at isolated spots while the general level of contamination in the groundwater is high. Table I. Contaminants in the groundwater Compound
1,1-Dichloroethane 1,2-Dichloroethane Trans-I ,2-Dichloroethene Dichloromethane I, I,I-Trichloroethane I, I ,-Dichloroethene Tetrachloroethene Trichloroethene Cis-I,2-Dichloroethene Vinylchloride dichlorobenzenes toluene
Abbreviation
Concentration (llg.l'I)
I,I-DCA 1,2-DCA trans-DCE DCM I,I,I-TCA I,I-DCE PCE TCE cis-DCE VC DCB TOL
20000 100
70 30000 70000 1500 20000 70000 20000 2000 3000 15000
The site was prepared for industrial use in 1951 by applying 2.5 m of dredging sludge from a nearby harbor. This was done by pumping the dredging sludge into the area causing the course material to settle near the outlet of the pipes and the fine materials further away. This process was repeated each time the pipes were lengthened to fill up the next section. As a result, clay-like layers are present in the first 2.5 m (Table 2). The lower layers represent the natural sequences caused by alternating periods of sedimentation and peat formation which is typical for the area where the site is located.
Stimulation of reducti ve dechlorination
107
Table 2. Soil profile at the site m below the soil surface
description
0-2.5
dredging sludge: very fine sand with low clay content light clay with a high organic matter content very fine sand with low clay content alternating layers of clay, sand and peat
2.5 - 5.0 5.0 - 6.0 6.0 - 18
The site was used from 1951 until 1972 for the activities indicated above. It appeared that the pollution is limited tot the top 2.5 m of the soil profile. The chlorinated solvents have in the meantime reached the surrounding premises of other firms leading to a total polluted surface of about 3,500 m 2. Since these firms are still active the total excavation of the polluted soil is not feasible. In addition the adjacent buildings are not sustained by concrete pillars on the deeper sand layers. Hence, excavation underneath and around the buildings is impossible and in-situ treatment is the only possible remediation solution. The high levels of VC at the site shows that natural dehalogenation is taking place already. However, this has not resulted in a significant reduction of the pollution during the 25 years of its existence. METHODS Column experiments Column experiments with both native and artificial groundwater were done to study possible strategies to enhance the full anaerobic dechlorination of PCE and TCE at the site. The columns were fed with either native groundwater amended with PCE, TCE and/or compost extract or artificial groundwater amended with PCE and defined electron donors. The native groundwater contained about 750 mg TOCn. Since sulfate is known to potentially inhibit reductive dehalogenation (Gibson and Suflita, 1986) the effect of the presence of sulfate was also evaluated.
soil column
mixing
chamber (synthetic)
gr oondwatar
Figure 1. Experimental set-up.
effluent
waste
108
P. 1. J. MIDDELDORP et al.
Glass columns (15 cm x 2.3 cm i.d.) were packed until approximately 5 cm from the top with soil taken from I metre depth of the polluted site at Maassluis, The Netherlands. The columns were percolated under upflow conditions at 20°C with medium, simulating the local groundwater composition (Fig. 1). PCE was added to the medium as a water saturated solution in a mixing chamber preceding the column, using a perfusor syringe pump (Perfusor VI, Braun Medical AG, Germany). This resulted in an influent concentration of approximately 50 J.1M in the columns with artificial groundwater. The columns with native groundwater were fed with a mixture of PCE and TCE at the same levels as found in the field (Table 1). The residence times in the columns were I day. Samples for analysis were taken from the influent and the effluent of the column with a glass syringe as described previously (Bosma et ai., 1988).
The artificial groundwater medium consisted of Na2HP04.2H20, 0.45 mM; KH 2P0 4, 0.15 mM; Na2S04' 0.06 mM; NaHC0 3 , I mM; NH 4CI, I mM; MgCI 2, 0.05 mM; MnCI 2, 0.02 mM; NaCI, 0.12 mM; demineralized water 1 litre. Optionally the following additions were made: Na2S04, I mM; CaCI 2, 6 mM, trace element solution (Holliger, 1992), 0.1 mUL. Oxygen was removed by boiling of the medium and purging with N 2 gas while cooling. Compost extract was prepared by stirring 450 g fresh compost with 1.5 L demineralized water for 3 days. Subsequently the extract was centrifuged (6000 rpm, 20 min.). The supernatant was then made up to 2 L with either groundwater or demineralized water. A concentrated salts solution was added to achieve the desired salts composition of the artificial groundwater. The compost extract contained about 800 mg TOctl. The media were continuously purged with N 2 gas throughout the experiments to maintain anoxic conditions. Sample preparation and analyses Samples from the influent and effluent of the columns were prepared by adding 6 mL sample to 13 mL serum bottles with viton stoppers (Maag Technik AG, Dtibendorf, Switzerland). The bottles were heated to 62°C in a water bath prior to analysis. 0.4 mL of the headspace was analyzed on Packard model 438A gas chromatograph equipped with a flame ionisation detector (AD) and a PLOT fused silica analytical column (Poraplot Q 16 J.1m, 25 m x 0.32 mm i.d.) from Chrompack (Middelburg, The Netherlands). Oven temperature conditions for the separation were: 50°C for 1 min, 39°/min to 210°C, total run length was 12 min. The concentrations of volatile fatty acids in the batch cultures were determined as follows: samples (I ml) were centrifuged (15 min., 13500 rpm), diluted twofold with 0.02 M H 2S0 4 and analyzed with an LKBHPLC system (PharmaciaILKB, Woerden, The Netherlands) equipped with a high performance (2150) pump and a differential refractometer (2142) detector. The samples (20 J.11) were applied to a Polyspher OA HY column (E. Merck), which was equilibrated with 0.01 M H 2S0 4 (flow rate 2 ml/min.). All quantifications were made using external standards. RESULTS AND DISCUSSION A series of column experiments was performed with both native and artificial groundwater as indicated in Table 3. The columns with native groundwater were fed with a mixture of PCE and TCE at the same levels as found in the field (Table I). The columns with artificial groundwater were fed with PCE at 50 J.1M. The breakthrough of PCE and the possible dechlorination products was followed during 100 days. The efficacy of various defined electron donors and compost extract were tested with artificial groundwater while only the addition of compost extract as electron donor was tested with the native groundwater. The potential inhibitory effect of sulfate was tested by leaving it out from the artificial medium after I month of operation.
Stimulation of reductive dechlorination
109
PCE was completely removed in all columns except the column fed with artificial groundwater without a«'lol'lion o'i e)ec'lroD oODDr 1'l' a'D)e ), 'IDe iDcomp)ele removID:m 'IDe £D)>>mJ) JeD w)i» memaJ)[») aJ)a so)Jate)s probably due to incomplete acclimation at the time of removing the sulfate, Sulfate does not seem to affect the reductive dechlorination in our columns markedly. In some cases the -If.' ",-.:.{{-u:.~ {'i'm1. ".oM, ",--M.-,:,Ym. {~~'i ~~Wfi, ".-0 'i~""Q{".';'O 18. ·tJ1ln'b.Yl\..tW~w;.~".ttt. ~t.~'m"im.".Wn,W(d..'y,~"J 'by improving the extent of dechlorination. This effect was observed in the columns fed with artificial groundwater amended with methanol, propionate, and lactate (Table 3). The extent of TeE formation from PCE increased from insignificant to 50% in the column fed with methanol, while cis-DCE appeared next to TCE in the column fed with propionate. However the improvements may also result from an acclimation that would also have occurred without leaving out the sulfate. WllfO"1l.
Tne ekc'lron donors 'lestea - acetate, met'naDo~, propionate, compos'l, ana ~acta'le - dean)' improve \'ne reductive dechlorination of PCE (Table 3). Interestingly, native groundwater amended with compost extract, was the only combination yielding the full dechlorination of PeE to ethene. With artificial groundwater, FCE wa'1> \ta'l)'1>1~tmeU \~ cis-OCE \'1) \~e ))te'1>etll:.e 1)1 ))t~))\I)WA\e, ~'AI:.\'A\e, 'AtlU I:.l)m))~'1>\ 'A'i\U \1) TeE \'i\ \.{\.e presence of acetate and methanol. These findings are in accordance with literature data that propionate and lactate better support reaucth'e aech}orination than acetate ami methanol DO, anD, that unDefined carbon sources are a prerequisite to obtain full reductive dechlorination of PCE and other chlorinated compounds (De Bruin et ai., 1992; Maymo-Gatell et al., 1997). Table 3. Final products of reductive dechlorination in the column experiments
Electron Groundwater donor
PCE Removal Sulfate"
artificial artificial artificial artificial artificial artificial artificial
none acetate acetate methanol methanol propionate propionate
+ +
artificial artificial artificial native native
lactate lactate compost none compost
+
(%)
50
Final product
>99 >99
TCE TCE TCE
+
<10
ND
+
>99 >99 >99
TCE TCE TCE/ cis-DCE cis-DCE cis-DCE cis-DCE cis-DCE ethene
+ + +
>99 >99 >99 >99 >99
Recoveryb (molar % of parent) 50 50 50 50 100 20/40 60 100 50 50 50
" Sulfate was left out from the influent after 1 month of column operation. b Since the mass balance was mostly incomplete, the recovery as end product of the parent compounds (sum ofPCE and TCE) added in the influent, are given.
CONCLUSIONS The field data suggest that full intrinsic dechlorination of PCE proceeds at the contaminated site albeit at very slow rates, which are insufficient to result in a rapid clean-up within the next few years.
110
P. J. J. MIDDELDORP etal.
The laboratory column results indicate the potential of a fast and complete reduction of PCE to ethene when compost extract is added to the native groundwater as an additional electron donor. Field tests to verify the effectiveness of compost extract as additive to stimulate intrinsic dechlorination of PCE are currently under way. ACKNOWLEDGMENTS This project was partially funded by the Dutch Research Program for Biotechnological In-Situ Remediation (NOBIS). The authors gratefully acknowledge Hugo van Buijsen and Geert Wijn for technical assistance. REFERENCES Bosma, T. N. P., Cottaar, F. H. M., Posthumus, M. A, Teunis. C. J .• Van Veldhuizen. A., Schraa, G. and Zehnder, A. J. B. (1994). Comparison of reductive dechlorination of hexachloro-I,3-butadiene in Rhine sediment and model systems with hydroxocobalamin. Environmelltal Science & Technology, 28,1124-1128. Bosma, T. N. P., Te Welscher, R. A. G., Smeenk, J. G. M. M., Schraa, G. and Zehnder, A. J. B. (1996). Biotransformation of organics in soil columns and an infiltration area. Ground Water, 34, 49-57. Bosma, T. N. P., Van der Meer, J. R., Schraa, G., Tros, M. E. and Zehnder, A J. B. (1988). Reductive dechlorination of all trichloro- and dichlorobenzene isomers. FEMS Microbiol. Ecol., 53, 223-229. De Bruin, W. P., Kotterman, M. J. J., Posthumus, M. A., Schraa, G. and Zehnder, A J. B. (1992). Complete biological reductive transformation of tetrachloroethene to ethane. Applied and Environmental Microbiology, 58, 1996-2000. Distefano, T. D., Gossett, J. M. and Zinder, S. H. (1991). Reductive Dechlorination of High Concentrations of Tetrachloroethene to Ethene by an Anaerobic Enrichment Culture in the Absence of Methanogenesis. Applied and Environmental Microbiology, 57, 2287-2292. Dolfing, J. and Tiedje, J. M. (1986). Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3chlorobenzoate. FEMS Microbiology Ecology, 38, 293-298. Fathepure, B. Z. and Vogel, T. M. (1991). Complete Degradation of Polychlorinated Hydrocarbons by a 2-Stage Biofilm Reactor. Applied and Environmental Microbiology, 57, 3418-3422. Gibson, S. A. and Sutlita, J. M. (1986). Extrapolation of biodegradation results to groundwater aquifers: reductive dehalogenation of aromatic compounds. Applied and Environmental Microbiology, 52, 681-688. Holliger, C. (1992). Reductive dehalogenation by anaerobic bacteria. : Agricultural University Wageningen, The Netherlands. Horowitz, A, Shelton, D. R., Cornell, C. P. and Tiedje, J. M. (1982). Anaerobic degradation of aromatic compounds in sediments and digested sludge. Dev [nd Microbiol, 23, 435-444. Maymo-Gatell, X., Chien, Y.-T., Gosset, J. M. and Zinder, S. H. (1997). Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science, 276,1568·1571. Middeldorp, P. J. M., Jaspers, M., Zehnder, A. J. B. and Schraa, G. (1996). Biotransformation of lX-, 13-, yo, and 6hexachlorocyclohexane under methanogenic conditions. Environmelltal Science & Technology, 30, 2345-2349. Mohn, W. W. and Tiedje, J. M. (1990). Strain DCB-l conserves energy for growth from reductive dechlorination coupled to formate oxidation. Archives of Microbiology, 153, 267-271. Mohn, W. W. and Tiedje, J. M. (1992). Microbial reductive dehalogenation. Microbiological Reviews, 56, 482-507. Verschueren, K. (1983). Handbook of environmental data on organic chemicals, 2 edn. New York: Van Nostrand Reinhold Company.
Pergamon
PI!: S0273-1223(98)00240-6
War. Sci. Tech. Vol. 37. No.8, pp. 105-110. 1998. © 1998 IA wQ. Published by Elsevier Science Ltd Printed in Great Britain. 0273-1223/98 $19'00 + 0'00
STIMULATION OF REDUCTIVE DECHLORINATION FOR IN SITU BIOREMEDIATION OF A SOIL CONTAMINATED WITH CHLORINATED ETHENES Peter J. M. Middeldorp*, Martine A. van Aalst**, Huub H. M. Rijnaarts*, Fons J. M. Stams*, Han F. de Kreuk***, Gosse Schraa* and TomN. P. Bosma** * Depanment of Microbiology,
Wageningen Agricultural University,
6703 CT Wageningen, The Netherlands
** TNO Institute for Environmental Sciences, Energy Research and Process Innovation, P.O. Box 342, 7300 AH Apeldoorn, The Netherlands *** BioSoil R&D B. V., 3341 U Hendrik Ido Ambacht, The Netherlands
ABSTRACT A soil from a fonner chemical redistribution company, contaminated with mainly chlorinated aliphatics, was studied for bioremediation purposes. Groundwater analyses revealed that the original pollutants, i.e. tetrachloroethene (PeE) and trichloroethene (TCE), were present at levels ranging from 2.3 to 122 mglL. Dichloroethene (DCE), vinylchloride (VC), ethene and ethane were also detected at significant concentrations although they had never been introduced to the soil. Relatively high concentrations of cisDCE as compared to trans-DCE and I,I-DCE indicated that a slow in situ biodegradation had taken place by reductive dechlorination. Laboratory experiments with flow-through soil columns were perfonned to detennine the optimal conditions for the enhancement of reductive dechlorination by the indigenous dechlorinating population. The addition of single electron donors to artificial groundwater resulted in the dechlorination of PCE to TCE and cis-DCE, whereas complete dechlorination to ethene was solely achieved with compost extract added to native groundwater. © 1998 IAWQ. Published by Elsevier Science Ltd
KEYWORDS Perchloroethene; trichloroethene; bioremediation; chlorinated solvents; BTEX; electron donor; compost. INTRODUCTION An important environmental problem is caused by the pollution of soils with PCE and TCE due to their wide spread use in dry cleaning and treatment of metal surfaces soil and groundwater pollution. The ex situ cleanup of these contaminated sites is costly and low-cost remediation strategies - e.g. involving biological methods - are urgently needed. 105
106
P. J. J. MIDDELDORP el al.
Anaerobic reductive dechlorination is proven to be an effective means for the detoxification of both aromatic (Bosma et at., 1988; Dolfing and Tiedje, 1986; Horowitz et al., 1982) and aliphatic (Bosma et at., 1994; Bosma et al., 1996; Middeldorp et at., 1996) halogenated compounds. Laboratory studies indicate that reductive dechlorination of PCE to less chlorinated compounds followed by aerobic treatment is a possible strategy for the clean-up of such sites (Fathepure and Vogel, 1991). However, the complete dechlorination of PCE and TCE via reductive dechlorination is also possible (De Bruin et at., 1992; Distefano et at., 1991) and may be simpler from the engineering point of view. However, the occurrence of the proven carcinogen VC (Verschueren, 1983) as an intermediate and possible end product of dechlorination needs attention. Earlier studies have showed the necessity of the presence of an electron donor to obtain the full dehalogenation of PCE under anaerobic conditions (De Bruin et at., 1992; Mohn and Tiedje, 1990; Mohn and Tiedje, 1992). The current study has been carried out at a heavily polluted site in the Netherlands where houses are going to be built. The aim of the present study was to find the most adequate electron donor for a complete reductive dechlorination of PCE - i.e. to ethene and/or ethane - under the conditions prevailing at the contaminated site. SITE DESCRIPTION The current study has been carried out at a heavily polluted site in the Netherlands where houses are going to be built. The site has been used by a firm redistributing a wide range of products from cleaning agents for metal finishing and for boiler maintenance to products for carbon removal from combustion engines. In addition metal surfaces were treated at the factory as well. These activities caused a soil pollution problem with a wide range of contaminants among which chlorinated solvents including PCE and TCE are the most important. Further contaminants are phenols, dichlorobenzenes, several aromatics such as benzene, toluene and xylenes (Table I) and non-chlorinated solvents with a f1ashpoint above 65°C. The top soil is contaminated at isolated spots while the general level of contamination in the groundwater is high. Table I. Contaminants in the groundwater Compound
1,1-Dichloroethane 1,2-Dichloroethane Trans-I ,2-Dichloroethene Dichloromethane I, I,I-Trichloroethane I, I ,-Dichloroethene Tetrachloroethene Trichloroethene Cis-I,2-Dichloroethene Vinylchloride dichlorobenzenes toluene
Abbreviation
Concentration (llg.l'I)
I,I-DCA 1,2-DCA trans-DCE DCM I,I,I-TCA I,I-DCE PCE TCE cis-DCE VC DCB TOL
20000 100
70 30000 70000 1500 20000 70000 20000 2000 3000 15000
The site was prepared for industrial use in 1951 by applying 2.5 m of dredging sludge from a nearby harbor. This was done by pumping the dredging sludge into the area causing the course material to settle near the outlet of the pipes and the fine materials further away. This process was repeated each time the pipes were lengthened to fill up the next section. As a result, clay-like layers are present in the first 2.5 m (Table 2). The lower layers represent the natural sequences caused by alternating periods of sedimentation and peat formation which is typical for the area where the site is located.
Stimulation of reducti ve dechlorination
107
Table 2. Soil profile at the site m below the soil surface
description
0-2.5
dredging sludge: very fine sand with low clay content light clay with a high organic matter content very fine sand with low clay content alternating layers of clay, sand and peat
2.5 - 5.0 5.0 - 6.0 6.0 - 18
The site was used from 1951 until 1972 for the activities indicated above. It appeared that the pollution is limited tot the top 2.5 m of the soil profile. The chlorinated solvents have in the meantime reached the surrounding premises of other firms leading to a total polluted surface of about 3,500 m 2. Since these firms are still active the total excavation of the polluted soil is not feasible. In addition the adjacent buildings are not sustained by concrete pillars on the deeper sand layers. Hence, excavation underneath and around the buildings is impossible and in-situ treatment is the only possible remediation solution. The high levels of VC at the site shows that natural dehalogenation is taking place already. However, this has not resulted in a significant reduction of the pollution during the 25 years of its existence. METHODS Column experiments Column experiments with both native and artificial groundwater were done to study possible strategies to enhance the full anaerobic dechlorination of PCE and TCE at the site. The columns were fed with either native groundwater amended with PCE, TCE and/or compost extract or artificial groundwater amended with PCE and defined electron donors. The native groundwater contained about 750 mg TOCn. Since sulfate is known to potentially inhibit reductive dehalogenation (Gibson and Suflita, 1986) the effect of the presence of sulfate was also evaluated.
soil column
mixing
chamber (synthetic)
gr oondwatar
Figure 1. Experimental set-up.
effluent
waste
108
P. 1. J. MIDDELDORP et al.
Glass columns (15 cm x 2.3 cm i.d.) were packed until approximately 5 cm from the top with soil taken from I metre depth of the polluted site at Maassluis, The Netherlands. The columns were percolated under upflow conditions at 20°C with medium, simulating the local groundwater composition (Fig. 1). PCE was added to the medium as a water saturated solution in a mixing chamber preceding the column, using a perfusor syringe pump (Perfusor VI, Braun Medical AG, Germany). This resulted in an influent concentration of approximately 50 J.1M in the columns with artificial groundwater. The columns with native groundwater were fed with a mixture of PCE and TCE at the same levels as found in the field (Table 1). The residence times in the columns were I day. Samples for analysis were taken from the influent and the effluent of the column with a glass syringe as described previously (Bosma et ai., 1988).
The artificial groundwater medium consisted of Na2HP04.2H20, 0.45 mM; KH 2P0 4, 0.15 mM; Na2S04' 0.06 mM; NaHC0 3 , I mM; NH 4CI, I mM; MgCI 2, 0.05 mM; MnCI 2, 0.02 mM; NaCI, 0.12 mM; demineralized water 1 litre. Optionally the following additions were made: Na2S04, I mM; CaCI 2, 6 mM, trace element solution (Holliger, 1992), 0.1 mUL. Oxygen was removed by boiling of the medium and purging with N 2 gas while cooling. Compost extract was prepared by stirring 450 g fresh compost with 1.5 L demineralized water for 3 days. Subsequently the extract was centrifuged (6000 rpm, 20 min.). The supernatant was then made up to 2 L with either groundwater or demineralized water. A concentrated salts solution was added to achieve the desired salts composition of the artificial groundwater. The compost extract contained about 800 mg TOctl. The media were continuously purged with N 2 gas throughout the experiments to maintain anoxic conditions. Sample preparation and analyses Samples from the influent and effluent of the columns were prepared by adding 6 mL sample to 13 mL serum bottles with viton stoppers (Maag Technik AG, Dtibendorf, Switzerland). The bottles were heated to 62°C in a water bath prior to analysis. 0.4 mL of the headspace was analyzed on Packard model 438A gas chromatograph equipped with a flame ionisation detector (AD) and a PLOT fused silica analytical column (Poraplot Q 16 J.1m, 25 m x 0.32 mm i.d.) from Chrompack (Middelburg, The Netherlands). Oven temperature conditions for the separation were: 50°C for 1 min, 39°/min to 210°C, total run length was 12 min. The concentrations of volatile fatty acids in the batch cultures were determined as follows: samples (I ml) were centrifuged (15 min., 13500 rpm), diluted twofold with 0.02 M H 2S0 4 and analyzed with an LKBHPLC system (PharmaciaILKB, Woerden, The Netherlands) equipped with a high performance (2150) pump and a differential refractometer (2142) detector. The samples (20 J.11) were applied to a Polyspher OA HY column (E. Merck), which was equilibrated with 0.01 M H 2S0 4 (flow rate 2 ml/min.). All quantifications were made using external standards. RESULTS AND DISCUSSION A series of column experiments was performed with both native and artificial groundwater as indicated in Table 3. The columns with native groundwater were fed with a mixture of PCE and TCE at the same levels as found in the field (Table I). The columns with artificial groundwater were fed with PCE at 50 J.1M. The breakthrough of PCE and the possible dechlorination products was followed during 100 days. The efficacy of various defined electron donors and compost extract were tested with artificial groundwater while only the addition of compost extract as electron donor was tested with the native groundwater. The potential inhibitory effect of sulfate was tested by leaving it out from the artificial medium after I month of operation.
Stimulation of reductive dechlorination
109
PCE was completely removed in all columns except the column fed with artificial groundwater without a«'lol'lion o'i e)ec'lroD oODDr 1'l' a'D)e ), 'IDe iDcomp)ele removID:m 'IDe £D)>>mJ) JeD w)i» memaJ)[») aJ)a so)Jate)s probably due to incomplete acclimation at the time of removing the sulfate, Sulfate does not seem to affect the reductive dechlorination in our columns markedly. In some cases the -If.' ",-.:.{{-u:.~ {'i'm1. ".oM, ",--M.-,:,Ym. {~~'i ~~Wfi, ".-0 'i~""Q{".';'O 18. ·tJ1ln'b.Yl\..tW~w;.~".ttt. ~t.~'m"im.".Wn,W(d..'y,~"J 'by improving the extent of dechlorination. This effect was observed in the columns fed with artificial groundwater amended with methanol, propionate, and lactate (Table 3). The extent of TeE formation from PCE increased from insignificant to 50% in the column fed with methanol, while cis-DCE appeared next to TCE in the column fed with propionate. However the improvements may also result from an acclimation that would also have occurred without leaving out the sulfate. WllfO"1l.
Tne ekc'lron donors 'lestea - acetate, met'naDo~, propionate, compos'l, ana ~acta'le - dean)' improve \'ne reductive dechlorination of PCE (Table 3). Interestingly, native groundwater amended with compost extract, was the only combination yielding the full dechlorination of PeE to ethene. With artificial groundwater, FCE wa'1> \ta'l)'1>1~tmeU \~ cis-OCE \'1) \~e ))te'1>etll:.e 1)1 ))t~))\I)WA\e, ~'AI:.\'A\e, 'AtlU I:.l)m))~'1>\ 'A'i\U \1) TeE \'i\ \.{\.e presence of acetate and methanol. These findings are in accordance with literature data that propionate and lactate better support reaucth'e aech}orination than acetate ami methanol DO, anD, that unDefined carbon sources are a prerequisite to obtain full reductive dechlorination of PCE and other chlorinated compounds (De Bruin et ai., 1992; Maymo-Gatell et al., 1997). Table 3. Final products of reductive dechlorination in the column experiments
Electron Groundwater donor
PCE Removal Sulfate"
artificial artificial artificial artificial artificial artificial artificial
none acetate acetate methanol methanol propionate propionate
+ +
artificial artificial artificial native native
lactate lactate compost none compost
+
(%)
50
Final product
>99 >99
TCE TCE TCE
+
<10
ND
+
>99 >99 >99
TCE TCE TCE/ cis-DCE cis-DCE cis-DCE cis-DCE cis-DCE ethene
+ + +
>99 >99 >99 >99 >99
Recoveryb (molar % of parent) 50 50 50 50 100 20/40 60 100 50 50 50
" Sulfate was left out from the influent after 1 month of column operation. b Since the mass balance was mostly incomplete, the recovery as end product of the parent compounds (sum ofPCE and TCE) added in the influent, are given.
CONCLUSIONS The field data suggest that full intrinsic dechlorination of PCE proceeds at the contaminated site albeit at very slow rates, which are insufficient to result in a rapid clean-up within the next few years.
110
P. J. J. MIDDELDORP etal.
The laboratory column results indicate the potential of a fast and complete reduction of PCE to ethene when compost extract is added to the native groundwater as an additional electron donor. Field tests to verify the effectiveness of compost extract as additive to stimulate intrinsic dechlorination of PCE are currently under way. ACKNOWLEDGMENTS This project was partially funded by the Dutch Research Program for Biotechnological In-Situ Remediation (NOBIS). The authors gratefully acknowledge Hugo van Buijsen and Geert Wijn for technical assistance. REFERENCES Bosma, T. N. P., Cottaar, F. H. M., Posthumus, M. A, Teunis. C. J .• Van Veldhuizen. A., Schraa, G. and Zehnder, A. J. B. (1994). Comparison of reductive dechlorination of hexachloro-I,3-butadiene in Rhine sediment and model systems with hydroxocobalamin. Environmelltal Science & Technology, 28,1124-1128. Bosma, T. N. P., Te Welscher, R. A. G., Smeenk, J. G. M. M., Schraa, G. and Zehnder, A. J. B. (1996). Biotransformation of organics in soil columns and an infiltration area. Ground Water, 34, 49-57. Bosma, T. N. P., Van der Meer, J. R., Schraa, G., Tros, M. E. and Zehnder, A J. B. (1988). Reductive dechlorination of all trichloro- and dichlorobenzene isomers. FEMS Microbiol. Ecol., 53, 223-229. De Bruin, W. P., Kotterman, M. J. J., Posthumus, M. A., Schraa, G. and Zehnder, A J. B. (1992). Complete biological reductive transformation of tetrachloroethene to ethane. Applied and Environmental Microbiology, 58, 1996-2000. Distefano, T. D., Gossett, J. M. and Zinder, S. H. (1991). Reductive Dechlorination of High Concentrations of Tetrachloroethene to Ethene by an Anaerobic Enrichment Culture in the Absence of Methanogenesis. Applied and Environmental Microbiology, 57, 2287-2292. Dolfing, J. and Tiedje, J. M. (1986). Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3chlorobenzoate. FEMS Microbiology Ecology, 38, 293-298. Fathepure, B. Z. and Vogel, T. M. (1991). Complete Degradation of Polychlorinated Hydrocarbons by a 2-Stage Biofilm Reactor. Applied and Environmental Microbiology, 57, 3418-3422. Gibson, S. A. and Sutlita, J. M. (1986). Extrapolation of biodegradation results to groundwater aquifers: reductive dehalogenation of aromatic compounds. Applied and Environmental Microbiology, 52, 681-688. Holliger, C. (1992). Reductive dehalogenation by anaerobic bacteria. : Agricultural University Wageningen, The Netherlands. Horowitz, A, Shelton, D. R., Cornell, C. P. and Tiedje, J. M. (1982). Anaerobic degradation of aromatic compounds in sediments and digested sludge. Dev [nd Microbiol, 23, 435-444. Maymo-Gatell, X., Chien, Y.-T., Gosset, J. M. and Zinder, S. H. (1997). Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science, 276,1568·1571. Middeldorp, P. J. M., Jaspers, M., Zehnder, A. J. B. and Schraa, G. (1996). Biotransformation of lX-, 13-, yo, and 6hexachlorocyclohexane under methanogenic conditions. Environmelltal Science & Technology, 30, 2345-2349. Mohn, W. W. and Tiedje, J. M. (1990). Strain DCB-l conserves energy for growth from reductive dechlorination coupled to formate oxidation. Archives of Microbiology, 153, 267-271. Mohn, W. W. and Tiedje, J. M. (1992). Microbial reductive dehalogenation. Microbiological Reviews, 56, 482-507. Verschueren, K. (1983). Handbook of environmental data on organic chemicals, 2 edn. New York: Van Nostrand Reinhold Company.