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Different abilities of eight mixed cultures of methane-oxidizing bacteria to degrade TCE
Wat. Res. Vol. 27, No. 2, pp, 215-224, 1993 Printed in Great Britain. All rights reserved
0043-13M/93 $5.00+0.00 Copyright © 1993 Pergamon Press Ltd
DIFFERENT ABILITIES OF EIGHT MIXED CULTURES OF METHANE-OXIDIZING BACTERIA TO D E G R A D E TCE K1M BROHOLM, THOMASH. CHgZS~SEN* and BJORNK. JENSEN Department of Environmental Engineering, Technical University of Denmark, Building 115, DK-2800 Lynghy, Denmark (First received November 1991; accepted in revised form August 1992)
Al~traet--The ability of eight mixed cultures of methane-oxidizing bacteria to degrade trichloroethylene (TCE) was examined in laboratory batch experiments. This is one of the first reported works studying TCE degradation by mixed cultures of methane-oxidizing bacteria at 10°C, a common temperature for soils and groundwaters. Only three of the eight mixed cultures were able to degrade TCE, or to degrade TCE fast enough to result in a significant removal of TCE within the experimental time, when the cultures used methane as growth substrate. The same three mixed cultures were able to degrade TCE when they oxidized methanol, but only for a limited time period of about 5 days. Several explanations for the discontinued degradation of TCE are given. An experiment carried out to re-activate the methane-oxidizing bacteria after 8 days of growth on methanol by adding methane did not immediately result in degradation of methane and TCE. During the first 10-15 days after the addition of methane a significant degradation of methane and a minor degradation of TCE were observed. This experiment revealed that the ability of mixed cultures of methane-oxidizing bacteria to degrade TCE varied significantly even though the cultures were grown under the same conditions. Key words--methane-oxidizing bacteria, chlorinated aliphatics, biodegradation, methane, methanol, trichloroethylene
INTRODUCTION
Chlorinated aliphatics are among the most widespread contaminants in subsurface environments due to their common use in industry for degreasing, dry cleaning and as solvents (Love and Eilers, 1982; Westrick et al., 1984). Research is currently been done to develop new methods for remediation of soil and groundwater contaminated with chlorinated aliphatics. Biological treatment of contaminated soil and groundwater, based on the ability of microorganisms to degrade these contaminants, is a promising method. Chlorinated aliphatics can be degraded under both aerobic and anaerobic (methanogenic) redox conditions. Under methanogeuic redox conditions chlorinated aliphatics are degraded by a reductive dechlorination where, as an example, tetrachloroethylene is degraded to TCE (Vogel et al., 1987). Unfortunately, the anaerobic degradation may result in an accumulation of less chlorinated aliphatic compounds, such as vinyl chloride (Vogel et al., 1987), which is a more hazardous compound than the parent compounds because of its carcinogenic properties (Windholz, 1983). Complete degradation of vinyl chloride to ethylene and carbon dioxide under anaerobic conditions has been observed only in a few studies (Vogel and McCarty, 1985; Freedman and Gossett, 1989). Under aerobic conditions the chlorinated aliphatics can be degraded cometabolically by special *Author to whom all correspondence should be addressed.
groups of bacteria, e.g. methane-oxidizing bacteria (Wilson and Wilson, 1985; Fogei et al., 1986; Strand and Shippert, 1986; Henson et al., 1988, 1989). Generally, the degradation rates increase with decreasing extent of chlorination, and tetrachloroethylene seems to be persistent (Fogel et aL, 1986; Henson et al., 1989). The pathways for aerobic cometabolic degradation of the chlorinated aliphatics have been investigated for TCE by Little et al. (1988). This pathway results in a complete mineralizaton of TCE to carbon dioxide and HCI via TCE-epoxide and other C r and C2-compounds. Some of these TCE degradation products have been proposed to inhibit TCE degradation under certain circumstances (Alvarez-Cohen and McCarty, 1991; Henry and Grbic-Galic, 1991; Oldenhuis et al., 1991). Despite this, aerobic degradation of TCE seems to be one of the most useful processes for remediation purposes. Methane-oxidizing bacteria are widespread in environments where both methane and oxygen are present, such as in transition zones between aerobic and anaerobic areas in soils or sediments (Hanson, 1980). The methane-oxidizing bacteria have been studied extensively during the last 20 years, and the pathway for degradation of methane (see Fig. 1) is well known (Dalton and Stifling, 1982). The first step, which is an oxidation of methane to methanol, is carried out by the enzyme methane monooxygenase, MMO, and requires an electron donor, such as NADH2, which is generated in the later oxidation steps. Methanol is oxidized to formaldehyde, which 215
216
IGM BROHOLM et al. o,
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Fig. 1. The pathway for methane degradation (Dalton and Stifling, 1982). is either assimilated into bacterial biomass or is further oxidized to formate and carbon dioxide generating N A D H 2 required for the initialoxidation of methane. M M O seems to be responsiblefor the initial oxidation of the chlorinated aliphatics(Fogel et aI., 1986). From the pathway shown in Fig. l it can be seen that methanol may be an alternative growth substrate (electrondonor and carbon source) for the methane-oxidizing bacteria, whereas formate only may serve as an alternativeelectron donor. Laboratory column and fieldexperiments investigating remediation schemes have revealed that it is difficultto achieve an effectivedegradation of T C E (and other chlorinatedaliphatics)in soiland groundwater (Semprini et aI., 1990; Broholm et aI., 1991). Therefore itisimperative to optimize the degradation process in order to develop a feasible remediation scheme. Several chemical factors influence the degradation of chlorinated aliphatics.The copper concentration seems to control whether methane-oxidizing bacteria express soluble or particulate M M O (Stanley et aI., 1983). During copper limitation,when the soluble form is expressed, higher degradation rates for the chlorinated aliphaticshave been observed compared to situationswith copper in excess (Oldcnhuis et aI., 1989; Tsien et aI., 1989). Studies on the interactions between T C E and methane degradation have revealed that the methane concentration has a negative effecton the degradation of T C E because of competition between T C E and methane for M M O (Lanzarone and McCarty, 1990; Broholm et aI., 1992; Oldenhuis et aL, 1991; Semprini et aI., 1991). The highest T C E degradation rateshave been observed by resting cells in the absence of additional methane. Unfortunately, resting cells have only a finiteT C E degradation capacity which may be due to toxicityof some of the T C E degradation products (AlvarczCohen and McCarty, 1991; Oldenhuis et aI., 1991). The use of alternative electron donors such as methanol and formate has been proposed to avoid the competition between T C E and methane. Methanol and formate are both miscible with water in contrast to methane which only has a limited solubility in water of 24 mg/l at 20°C 0Vindholz, 1983). Formate has been used successfully as an electron donor to enhance the T C E degradation by resting cellsfor shorter periods of time (from a few
hours to a few days) (Oldenhuis et al., 1989; AlvarezCohen and McCarty, 1991; Semprini et al., 1991), whereas the use of methanol so far has resulted in differing results (Janssen et al., 1988; Oldenhuis et al., 1989). Henry and Grbic-Galic (1990) have shown that the TCE degradation rates for different mixed and pure cultures of methane-oxidizing bacteria grown in slightly different mineral media varied by more than a factor of 10. Besides the above-mentioned chemical factors affecting the degradation of chlorinated aliphatics, the nature of the actual microbial consortia may also be an important factor. If highly efficient cultures could be identified they might be used as inoculum for rvmediation purposes. The reported experiments on TCE degradation involving mixed and pure cultures of methane-oxidizing bacteria apparently reveal large variations in TCE degradation rates, but so far only one investigation of the diversity among mixed cultures of methane-oxidizing bacteria to degrade TCE has been reported (Henry and Grbic-Galic, 1991). That investigation revealed large differences between TCE degradation rates for resting cells of three mixed cultures. Generally, the reported experiments have been carried out at temperatures between 20 and 35°C, which are much higher than the natural groundwater temperature. Therefore, the degradation of chlorinated afiphatics by methane-oxidizing bacteria has to be studied at groundwater temperatures in order to make the obtained results more applicable for natural groundwater and soil systems. This paper reports the results of laboratory experiments carried out at 10°C investigating the ability of eight mixed cultures of methane-oxidizing bacteria to degrade TCE. Mixed cultures, in contrast to pure cultures, are investigated since an initially amended pure culture is likely to develop into a mixed culture in non-sterile environments, such as soils and groundwaters, being supplied with methane and oxygen. Furthermore, it is more difficult to obtain pure cultures than mixed cultures. The inocula for the mixed cultures were collected at waterworks that treat methane-containing groundwater for public water supply. The aeration and filtration units of such waterworks contain relatively stable transition zones between a reduced, methane-rich environment (anaerobic groundwater) and an aerobic environment,
TCE degradation by mixed cultures of methane-oxidizing bacteria creating ideal conditions for m e t h a n e - o x i d i z i n g bacteria. This study covers three experimental series. T h e first series of experiments focused o n t h e ability o f the eight mixed cultures to degrade T C E d u r i n g m e t h a n e oxidation, while the second series o f experiments investigated the ability of the same m i x e d cultures to degrade T C E during methanol oxidation. T h e third experimental series investigated the effect o f alternating methane a n d methanol additions u s i n g m e t h a n e for enzyme induction and m e t h a n o l for bacterial growth. MATERIALS AND METHODS
The mixed cultures of methane-oxidizing bacteria Eight mixed cultures of methane-oxidizing bacteria were obtained from seven different Danish waterworks that treat groundwater containing methane. Material taken from the sand and/or the aeration filter was used as inoculum. The inocuia for the Cultures 1, 2, 3 and 5A originated from sand filters consisting of sand or gravel, while the inocula for the Cultures 4, 5C, 6 and 7 originated from aeration filter sludge. About 50 mi of each inoculum was mixed with 150 ml of nutrient solution in 1 liter glass bottles. 20 ml of methane was added to the headspace resulting in an initial methane concentration of 2.5% (vol/vol) in headspace and 0.75 mg/l in liquid phase. The bottles were incubated in the dark at 10°C with magnetic stirring. Weekly, about 50 ml of each culture was diluted with 150 ml of nutrient solution and new methane was added. Initially, some of the inocula consisted of solids, but after subsequent dilutions the cultures only consisted of liquid. After about 2 months significant methane consumption and bacterial growth was observed, and the cultures were then used for the batch test experiments. The nutrient solution contained the following macroand micronutrients per liter distilled water: 5.8rag of Na2HPO4"2H20, 3.8mg of NH4C1, 147rag of CaCl 2, 122mg of NaNO 3, 100rag of NaHCO3, 9.4rag of KC1, 0.4mg of FeCI3"6H20, 50rag of MgSO4"7H20, 10/~g of MnCI2"4H20, 12/~g of ZnSO4.7H20, 6/~g of CuSO4" 5H20, 6/zg of CoCI 2.6H20 , 5.5 #g of Na2MoO 4.2H20, 2/~g of Na2B4OT.10H20. The pH of the solution was adjusted to 7. The concentration of some of the individual ions is shown in Table 1. Water samples for characterization of the original environments of the cultures were taken from the same place as the inocula except for Cultures 4 and 5C, where samples
217
were taken from the sand filters. Temperature, O2-concentration and pH were measured on site, whereas the other parameters were determined in the laboratory on water samples preserved in accordance with the analysis. Table 1 shows the chemical characteristics of the water samples representing the original environment of the studied cultures. The chemical composition of the water samples was quite similar except for nitrate, ammonium, sulfate, magnesium and iron, where a difference of more than a factor of ten between the water samples was observed. Also for methane a difference of a factor of ten was observed, probably because Samples 6 and 7 were taken at the aeration filters where the groundwater still contains high concentration of methane due to a yet incomplete stripping of methane. The chemical composition of the nutrient solution was similar to the water samples except that the nutrient solution contains high concentrations of nitrate and phosphate, two essential nutrients for the bacteria and low iron concentration.
Description of the batch experiments All the experiments were carried out in l l 7 m l glass bottles equipped with Miniert valves (DYNATECH Precision Sampling Corporation, Baton Rouge, La), which enabled frequent sampling from the bottles. Each bottle contained 10 ml inoculum and 10 ml nutrient solution. The inocula in all the experiments were mixed cultures of bacteria grown with methane as the sole energy and carbon source. The control bottles contained 20 ml nutrient solution and 0.3 g sodium azide (NAN3) per liter to prevent any bacterial growth. The bottles were incubated in a rotating box (10 rpm) in the dark at 10°C, the groundwater temperature in Denmark (see Table 1). In the TCE degradation experiment with methane as growth substrate the batches contained TCE at initial liquid concentrations of 500 pg/l and 0.17 mmol methane. Methane partitioned between the liquid phase and the headspace resulting in an initial concentration of methane in the liquid phase of 0.075 retool/1 (1.2 mg/l). Methane was added to the headspace with a syringe. 50/~1 of a stock solution of TCE, prepared by dissolving a known volume of TCE in a nutrient solution, was added to each batch. In the TCE degradation experiment with methanol as growth substrate, the batches contained TCE at initial liquid concentrations of 500 ~g/l and 0.17 mmol methanol. The initial methanol concentration in the liquid phase was 8.5mmol/l (272mg/1). Methanol was dissolved in the nutrient solution before it was added. In the experimental series with alternating methanol and methane addition the initial liquid concentration of TCE was 500 ~g/i and the initial concentration of methanol in the liquid phase 8.5 retool/1. In this experiment both methane
Table 1. Chemical characterization of the water samples taken at the waterworks and the nutrient solution. The water samples from Waterworks 1, 2, 3, 4 and 5 were taken from the sand filter and 6 and 7 were from the aeration filter Nutrient Waterworks 1 2 3 4 5 6 7 solution Temperature (°C) 9.3 11.0 9.6 11.4 10.0 10.2 NA NA pH 7.27 7.78 7.23 8.16 7.32 7.77 7.76 7.0 Oxygen (mg/I) 9.9 10.0 9.0 9.3 9.5 9.4 10.1 NA Methane (rag/I) 0.23 0.98 0.02 0.87 0.04 1.08 4.00 0 TAL (mequlv/I)* 5.5 5.8 5.4 7.9 4.5 5.4 6.9 1.0~ TOC (rag C/I)t 2.1 3.9 2.6 5.7 1.3 4.2 4.9 0 Nitrate (rag N/I) 0.02 0.04 0.16 0.15 0.13 0.07 0.01 20 Ammonium (mg N/I) 0.70 0.83 0.18 !.8 0.17 0.56 1.2 1.0 Sulfate (rag/I) 13 7 63 13 52 i2 8 20 Phosphate (rag/l) 0.19 0.22 0.07 0.14 0.06 0.09 0.26 3. I Calcium (rag/I) 99 91 127 ! 15 116 122 98 57 Magnesium (rag/I) 18 22 20 66 17 18 23 4.9 Iron (mg/I) 2.2 2.3 1.4 0.3 1.2 1.2 1.8 0.09 Manganese (mg/l) 0.06 0.06 0.08 <0.02 0.06 <0.02 0.08 0.003 Copper ~g/I) 1.4 0.8 1.0 5.0 0.8 0.6 1.2 1.5 *TAL = total alkalinity; tTOC = total organic carbon; :~NAffinot analyzed; ~calculated.
218
KIM BROHOLMe l
and methanol were added during the experiment. 6.5 pl pure methanol (0.16 mmol)was added with a syringe after most of the methanol in the batches had been oxidized. 1 m] pure methane (0.043 mmol) was added to the batches after 8 days of operation. Assuming all methane was in the liquid phase the initial molar methane concentration was 2.25 retool/l,
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219
TCE degradation by mixed cultures of methane-oxidizing bacteria
in the control experiments within 30 days was about 10% of the initial amount of TCE. The molar concentration of methane shown in Figs 2 and 4 is calculated as if all the methane at the time of sampling was in the liquid phase.
initial biomass concentration in water samples at the start of the experiments was measured as protein. The relative TCE concentration shown in Figs 2 and 3 is calculated as (C/Co)/(C~u~/C~u~.o) where C and Co=u~ are liquid TCE concentrations at the time of sampling in the degradation batches and in the control batches, Co and C~o~,o are initial 5quld TCE concentrations in the degradation batches and in the control batches. The loss of TCE
Chemicals Methane >99.95% pure, was purchased from A/S Dansk Ilt & Brintfabrik, Denmark. Methanol, >99.7%,
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Fig. 3. The degradation of TCE and methanol for four mixed cultures (Cultures 3, 4, 5A and 7) of methane-oxidizing bacteria obtained from waterworks treating groundwater containing methane. The individual results of three batches and the average (dashed lines) are shown in the figure. WR 2"/r2---c
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Fig. 4. The degradation of methanol and methane for one mixed culture (Culture 7) of methane-oxidizing bacteria and a control in an experiment where the bacteria grew only on methanol the first 8 days. At day 8 methane and methanol were added to the batches, and further methanol was added at day 15. The individual results of three batches and the average (dashed lines) are shown in the figure. The molar concentration of methane was calculated as if all methane at the sampling time was in the liquid phase. Protein measurements were performed on fiquid samples to which 3 M trichloroacetic acid solution was added to kill the bacteria before storing at 4°C. The samples were centrifuged for 20rain at 4000 rpm in a Heraens Christ Labofuge III. The supernatant was discarded carefully and 0.66 N sodium hydroxide solution was added to the pellet Analytical procedures to lyse the bacteria. After incubation for 2 days at 35°C and staining with Coomamie brilliant blue G-250 the protein Methane, methanol and TCE were analyzed on a DANI 8520 gas chromatograph equipped with a flame ionization content was measured spectropbotometrically at 595 um by detector for methane and methanol and an electron capture a Bechman DB GT grating spectrophotometer. The preparation of Coomassie brilliant blue G-250 and the protein detector for TCE. Chromatograph separation was obtained using a 30 m long penetrated open tubular fused silica standards are described by Bradford (1976). pH was measured with a Microprocessor pH Meter column (0.53 mm i.d.) coated with CP-SIL5 cross bounded. pH 196, WTW, and oxygen and temperature were measured The gas chromatograph was operated isothermally at a with a Microprocessor Oximeter oxi 196, WTW. column temperature of 50°C for methane and TCE, and Calcium, magnesium, manganese and iron were analyzed I05°C for methanol, and detector temperature of 275°C for both detectors. The carrier gas was nitrogen. Data acqui- by means of flame atomic absorption spectrophotometry on sition and integrations were performed on a MAXIMA a Perkin-Elmer 370. Copper was measured on an inductively coupled plasma mass spectrometer (IPC-MS) Chromatography Workstation. Air samples for methane were injected with a gas-tight (Perkin-Elmer 5000). Total organic carbon (TOC) was syringe and the concentrations were determined by compari- measured on a Dohrmann DC 180. Nitrate, ammonium, sulphate and phosphate were anason with a standard curve. Methane in water was measured on samples taken with a syringe and injected into a vial with lyzed on a Technicon AutoAnalyser. Nitrate was detera vacuum. After methane had equilibrated between the mined by use of a hydraziue reduction method based on a water and the headepace, the concentration of methane in modification of a procedure of Kamphake et al. 0967). the headspace was measured as previously described and the P-mmonium WaS determined by use of a phenate method original methane concentration in the water sample was described in APHA (1989), sulfate by use of an automated methylthymol blue method described in ALPHA (1989) and calculated. TCE was measured on air samples which were injected phosphate by use of an ascorbic acid method described in directly and the concentrations were determined by com- APHA (1989). Total alkalinity (TAL) was measured on a Radiometer parison with standards made in pentane. The concentration of TCE in the liquid phase was calculated from the Autoburette ABU 11 equipped with a Radiometer Research measured concentration in the headspace tufing a dimension- pH Meter PHM 64. The rumples were titrated with H2SO4 less Henry's law constant of 0.17 at 10°C (Gossett, 1987). to pH 4.5.
sodium azide, >99%, and TCE, >99.5%, were purchased from Merck, Darmstadt, Germany. Coomassie brilliant blue G-250 and bovine albumin for protein standards were purchased from Sigma Chemical Co., St Louis, Mo., U.S.A. The nutrients were of reagent grade quality.
TCE degradation by mixed cultures of methane-oxidizing bacteria RESULTS
The mixed cultures used as inocula in all the experiments used methane as the sole energy and carbon source. They are referred to as mixed cultures of methane-oxidizing bacteria even though they consist of methane-oxidizing bacteria and other bacteria. Most methane-oxidizing bacteria are capable of using methanol as growth substrate as well as methane (Whittenbury et al., 1970). The mixed cultures probably contained methane-oxidizing bacteria (have MMO, are capable of using methane and methanol as growth substrate and may be able to degrade TCE), methanol-oxidizing bacteria (do not have MMO, are able to use methanol as growth substrate and probably not able to degrade TCE) and other bacteria.
Degradation of TCE with methane as growth substrate Significant TCE degradation was observed for only three of the eight mixed cultures (Cultures 4, 5A and 7), when methane was added as growth substrate. The TCE and methane degradation curves for these three mixed cultures and for Culture 3, which was unable to degrade TCE, are shown in Fig. 2. At the end of the experiments after 30 days the average observed degradation of TCE was between 28 and 55% of the initial amount of TCE for the three mixed cultures (Cultures 4, 5A and 7) (Fig. 2). The fact that no TCE degradation was observed within 30 days for the other five mixed cultures (Cultures 1, 2, 3, 5C and 6) does not necessarily mean that these cultures are unable to degrade TCE. The TCE degradation rates for the bacteria in these mixed cultures may be so low that they would not result in significant removal of TCE within the experimental time. The results of the TCE degradation experiments with methane as growth substrate are summarized in Table 2, where the time period for 50% degradation of the initial amount of methane for all eight mixed cultures is shown together with the ability of the mixed cultures to degrade TCE. A mixed culture was considered to degrade TCE if more than 10% of the initial amount of TCE was removed within the experimental time. The time period until 50% of the initial amount of methane was degraded varied between 7 and 17 days for the eight mixed cultures (Table 2). All the cultures except Culture 5C de-
221
graded more than 90% of the initially added methane within 30 days. The three mixed cultures which degraded a significant amount o f TCE were not the fastest methane degrading cultures. The initial concentration of biomass in each experiment measured as protein is also shown in Table 2. The initial concentration o f protein in the experiments was between 2.5 and 14.4mg]l [equal to 10~-6 × 10~ bacteria/ml, using conversion factors of 0.5mg protein/rag cell dry weight (Nester et al., 1983), and 2 x 109bacteria]mg cell dry weight (Balkwill et al., 1988)]. The protein concentration is not a specific measurement of the number of methane-oxidizing bacteria in the mixed cultures, but only gives the total amount of bacteria in the mixed cultures. It is obvious that the methane-oxidizing bacteria in the mixed cultures are responsible for the observed degradation of methane and TCE.
Degradation of TCE with methanol as growth substrate The results of the TCE degradation experiments for four of the eight mixed cultures (Cultures 3, 4, 5A and 7) of bacteria with methanol as growth substrate are shown in Fig. 3. The mixed cultures used as inoculum in these experiments grew with methane as the only carbon and energy source before the start of the experiment. An average TCE degradation of 22-40% of the initial amount of TCE was observed for three mixed cultures (Cultures 4, 5A and 7) (Fig. 3). The mixed cultures for which a significant degradation of TCE was observed when methanol was added as growth substrate were the same mixed cultures for which a significant TCE degradation was observed when methane was added as growth substrate. The degradation of TCE stopped after about 5 days even though the mixed cultures continued to degrade methanol. At the time when the bacteria had degraded all the initially added methanol, the batches were re-fed with methanol (data not shown in Fig. 3). The results of the TCE degradation experiments with methanol as growth substrate are summarized in Table 2, where the time period for 50% degradation of the initial amount of methanol for all eight mixed cultures is shown together with the ability of the mixed cultures to degrade TCE when the
Table 2. Summaryof the TCE degradationexperimentsfor 8 mixed culturesof methane-oxidizingbacteriawith methane or methanol as growth substrate. The time period in days for 50% degradationof the growth substrateis shown together with the mixedculturesabifities for degradingTCE with methane and methanol.Degradationof TCE was consideredsignificantif more than 10"/, of TCE was removed. The initial protein concentrationin the experimentsis also shown Culture number 1" 2* 3* 4t 5A* 5Ct 6t 7t Initial protein cone. (rag/I) 2.5 3.6 14.4 10.2 Time period for 50% degradationof methane (days) 12 9 7 11 TCE degradation with methane + Time period for 50% degradationof methanol (days) 6 4 2 3 TCE degradationwith methanol + *The mixed culture originated from sand filtersat a groundwater-lmsedwaterworks. tThe mixed culture orisinated from aeration filtersat a groundwater-basedwaterworks.
9.3 12 + 4 +
5.3 17 3 --
13.6 10 3 --
10.1 10 + 6 +
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Kn~ BROHOLMet al.
mixed cultures used methanol as growth substrate. Methanol degradation was observed for all eight mixed cultures (Table 2). The time period for 50% degradation of the initial amount of methanol varied between 2 and 6 days (Table 2). The initial concentration of biomass in each experiment measured as protein was the same as in the degradation experiments where methane was added as growth substrate (Table 2). It is impossible to distinguish between the methanol degradation by methane-oxidizing bacteria and the methanol degradation by methanol-oxidizing bacteria based on the methods used in this study. Alternating methane and methanol addition
These experiments were carried out in a similar way as the experiments with only methanol as growth substrate except that methane was added to the batches at day 8. Before the experiment (day 0) the mixed cultures (Cultures 4, 5A and 7) had only grown with methane as the sole growth substrate. From day 0 to day 8 the bacteria only grew with methanol, whereas both methane and methanol were present from day 8 and to the end of the experiments. TCE was added to the experiments at day 0. The removal of methane and methanol for Culture 7 and a control experiment is shown in Fig. 4. The results for Cultures 4 and 5A are similar to the results for Culture 7. A fast methanol degradation was observed during the whole experiment. At the time methane was added at day 8 and until day 19 only a minor methane and no TCE degradation was observed. After day 19 a significant methane and a minor TCE degradation was observed. The reason why methane was added at day 8 was that the experiment with methanol as growth substrate revealed that the mixed cultures did not retain the ability to degrade TCE while using methanol as growth substrate. If the methane-oxidizing bacteria lost the ability to degrade TCE because they did not produce MMO while oxidizing methanol, then the addition of methane at day 8 should result in a production of MMO followed by a degradation of TCE. DISCUSSION
Eight mixed cultures were obtained from different inocula, but were grown at the same growth conditions: at 10°C, with the same nutrient solution, and with methane as the sole carbon and energy source. TCE degradation experiments with methane as growth substrate showed that only three of the eight mixed cultures were able to degrade TCE or able to degrade TCE fast enough to result in a significant TCE degradation within 30 days. The three mixed cultures (Cultures 4, 5A and 7) which were able to degrade TCE were not the fastest methane-degrading cultures (Table 2). As the mixed cultures were grown at similar conditions the observed differences with
respect to TCE degradation between the methaneoxidizing bacteria in the mixed cultures are probably due to different inherent abilities of the methaneoxidizing bacteria to degrade TCE. The same three mixed cultures, which were able to degrade TCE when the mixed cultures used methane as growth substrate, were able to degrade TCE when the cultures used methanol as growth substrate. Unfortunately, the TCE degradation occurred only within the first 5 days even though methanol was still present in the batches at that time. Obviously, methanol-degrading bacteria were present in the batches during the whole experiments, whereas methane-oxidizing bacteria was present at the start of the experiments and may not have been active for more than 5 days. This indicates that it was the methane-oxidizing bacteria in the mixed cultures which were responsible for the observed degradation of TCE in the experiments with methanol as growth substrate. At least three explanations why the methane-oxidizing bacteria did not continue to degrade TCE with methanol as growth substrate instead of methane are given below. One explanation is that the degradation of TCE results in production of TCE-epoxide or other intermediate products, which may be toxic to the methane-oxidizing bacteria. The experiments, reported in the literature, showing inhibition of resting cells of methane-oxidizing bacteria by TCE degradation products have been carried out with relatively high TCE concentration and with relatively high biomass to achieve a fast degradation of TCE and a fast inactivation of the cells (few hours) (Alvarez-Cohen and McCarty, 1991; Oldenhuis et al., 1991). In contrast to this, our experiments were carried out with relatively low TCE concentration and relatively low biomass resulting in a slow TCE degradation and a slow inactivation of the cells (5 days). Our observations have been confirmed at field conditions by Semprini et al. (1991) who successfully used methanol as alternative substrate to support TCE degradation for 60 h. The field experiment was carried out with low biomass concentration and low TCE concentration, but the time period where the bacteria used methanol was not sufficiently long to show whether inactivation of the cells occurred. Degradation studies with c/s- and trans-l,2-dichloroethylene (c1,2-DCE and t-I,2-DCE) by mixed cultures of methane-oxidizing bacteria in the presence of methane have revealed accumulation of c- and t-l,2DCE-epoxide outside the cells, which shows that c-I,2-DCE and t-I,2-DCE degradation products may not bind to the cells at these growth conditions (.Ianssen et al., 1988; Strandberg et al., 1989). This is not necessarily valid also for TCE. The half-lives for chemical conversion of TCE-epoxide of 1.3min (Kline et al., 1978), for c-l,2-DCE-epoxide of about 72h and for t-l,2-DCE-epoxide of about 30h (Janssen et al., 1988), make it more likely to observe accumulation of the epoxide of c- or t-I,2-DCE than
TCE degradation by mixed cultures of methane-oxidizing bacteria of TCE. This indicates that the TCE degradation products may be accessible for further conversion, and for this reason, the time period until total inactivation of the cells becomes an important factor. In our experiments with relatively slow TCE degradation the TCE degradation products may have sufficient time to undergo further conversion, which makes an inhibition of the cells by the degradation products more unlikely than in experiments with a fast TCE degradation. However, any final conclusion whether or not the observed inactivation of the cells is due to inhibitory effect by TCE degradation products cannot be made. A second explanation is that the methane-oxidizing bacteria in the mixed cultures do not get the required energy (NADH2) for the initial oxidation of TCE. When methane-oxidizing bacteria oxidize methanol they produce N A D H 2 which theoretically could supply MMO with the required energy for TCE oxidation. Therefore it is unlikely that the discontinued TCE degradation was due to lack of energy. The third explanation is that the methane-oxidizing bacteria in the mixed cultures do not produce MMO in absence of methane. The observed TCE degradation in our experiments which stopped after 5 days may be due to some MMO the methane-oxidizing bacteria already had produced when the bacteria grew on methane before the start of the experiments. The literature contains contradictory results about the induction of MMO. A pure culture of methaneoxidizing bacteria (Methylosinus trichosporium OB3b) grown on methanol has been shown to retain the activity of MMO for 9 months (Best and Higgins, 1981). Therefore, it is possible for some methaneoxidizing bacteria to continue degrading TCE using methanol as growth substrate, if the TCE degradation products do not inhibit the bacteria. However, the methane-oxidizing bacteria in the mixed cultures in our experiments do not seem to retain their MMO activity in absence of methane assuming that the bacteria were not inhibited by TCE degradation products or did not lack energy for the continuing degradation of TCE. The result of the experiment with alternating methane and methanol addition showed no immediate methane degradation at the time methane was added after 8 days where the bacteria oxidized methanol (Fig. 4). It took about 10-15 days before a significant methane degradation and a minor TCE degradation was observed. The fact that no immediate methane degradation was observed at day 8 showed that either the methane-oxidizing bacteria were not present at that time or were not re-activated immediately. After 10-15 days methane degradation was observed revealing that methane-oxidizing bacteria were also present at day 8 but the number of methane-oxidizing bacteria may be too small to result in a significant methane degradation. No degradation of T e E was observed for Cultures 4, 5A and 7 within the first 5 days of this experiment,
223
which is in contrast to previous experiments. AlvarezCohen and McCarty (1991) have shown that the amount of TCE degraded by resting cells depends on several factors, e.g. the time period the cells have been in absence of methane. This may explain why the same cultures in one case degrade a certain amount of TCE for 5 days and in another case do not degrade a significant amount of TCE. The fact that no or very little TCE degradation occurred within the first 8 days of this experiment eliminates the explanation that the methane-oxidizing bacteria at day 8 were inactivated by TCE degradation products. CONCLUSION Eight mixed cultures of methane-oxidizing bacteria grown in similar conditions showed different abilities to degrade TCE when using either methane or methanol as growth substrate. For only three mixed cultures (Cultures 4, 5A and 7) was a significant TCE degradation observed within the experimental time when the mixed cultures used methane as growth substrate. When the same three mixed cultures were oxidizing methanol they were able to degrade TCE, but the ability was present only during the first 5 days of the experiments. It was not possible to re-activate the methane-oxidizing bacteria after 8 days of methanol oxidation by adding methane. The observation that some of the mixed cultures did not degrade TCE (or degraded TCE very slowly) and some degraded TCE relatively fast under similar growth conditions is an important observation dealing with bioremediation of soil or groundwater. If an indigenous population of methane-oxidizing bacteria is present in the soil and/or groundwater, and stimulated by the addition of methane and oxygen, then the required time period for /n situ bioremediation depends on what kind of methane-oxidizing bacteria are present at the actual site. In order to inoculate soils and/or groundwaters or on-site treatment plants with methane-oxidizing bacteria it is important to inoculate with a culture, which is able to degrade TCE (or other chlorinated aliphatics) fast at the actual growth conditions. Acknowledgements--The financial support from the National Agency of Environmental Protection in Denmark is gratefully acknowledged. We thank Michael I.~fvall, Department of Environmental Engineering, Technical University of Denmark, for help with the experiments and the analysis. REFERIgNCI~
Aivarez-Cohen L. and McCarty P. L. (1991) TCE transformation by a mixed methanotrophic cuiture-effects of toxicity, aeration and reductant supply. Appl. envir. Microbioi. 57, 228-235. APHA (1989) Standard Methods for the Examination of Water and Wastewater, 17th edition. American Public Health Association, Washington, D.C. Balkwill D. L., Leach F. R., Wilson J. T., McNabb J. F. and White D. C. (1988) Equivalence of microbial biomass
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measures based on membrane lipid and cell wall components, adenosine triphosphate, and direct counts in subsurface aquifer sediments. Microb. Ecol. 16, 73-84. Best D..I. and I-Iiggins I. J. (1981) Methane-oxidizing activity and membrane morphology in a methanol-grown obligate methanotroph Methylosinus trichosporium OB3b. J. gen. Microbiol. 125, 73-84. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Analyt. Biochem. 72, 248-254. Brobolm K., Christensen T. H. and Jonson B. K. (1991) Laboratory feasibility studies on biological in-situ treatment of a sandy soil contaminated with chlorinated aliphatics. Envir. TeclmoL 12, 279-289. Broholm K., Christensen T. H. and Jonson B. K. (1992) Modelling TCE degradation by a mixed culture of methane-oxidizing bacteria. Wat. Res. 26, 1177-I 185. Dalton H. and Stirring D. I. (1982) Co-metabolism. Phil. Trans. R. Soc. Lond. 297, 481-496. Fogel M. M., Taddeo A. R. and Fogel S. (1986) Biodegradation of chlorinated ethenes by a methane-utilizing mixed culture. Appl. envir. Microbiol. 51, 720-724. Freedman D. L. and Gossett J. M. (1989) Biological reductive dechlorination of tetrachloroethylene and trichloroethylene to ethylene under methanogenic conditions. Appl. envir. Microbiol. 55, 2144-2151. Gossett J. M. (1987) Measurement of Henry's law constant for C, and C2 chlorinated hydrocarbons. Envir. Sci. Technol. 21, 202-208. Hanson R. S. (1980) Ecology and diversity of methylotrophlc organisms. Adv. appl. Microbiol. 26, 3--39. Henry S. M. and Grbic-Galic D. (1990) Effect of mineral media on trichloroethylene oxidation by aquifer methanotrophs. Microb. Ecol. 20, 151-169. Henry S. M. and Grbic-Galic D. (1991) Influence of endogenous and exogenous electron donors and trichloroethylene oxidation toxicity on trichloroethylene oxidation by methanotrophlc cultures from a groundwater aquifer. Appl. envir. Microbiol. 57, 236-244. Henson J. M., Yates M. V. and Cochran J. W. (1989) Metabolism of chlorinated methanes, ethanes, and ethylenes by a mixed bacterial culture growing on methane. J. Ind. Microbiol. 4, 29-36. Henson J. M., Yates M. V., Cochran J. W. and Shackleford D. L. (1988) Microbial removal of halogenated methanes, ethanes, and ethylenes in an aerobic soil exposed to methane. FEMS Microbiol. EcoL 53, 193-201. Janssen D. B., Grobben G., Hoektra R., Oldenhuis R. and Witholt B. (1988) Degradation of trans-l,2-dichloroethene by mixed and pure cultures of methanotrophic bacteria. Appl. Microbiol. Biotechnol. 54, 951-956. Kampbake L. J., Hannah S. A. and Cohen J. M. (1967) Automated analysis for nitrate by hydrazine reduction. War. Resour. Res. 1, 205-216. Kline S. A., Solomon J. J. and Duuren B. L. V. (1978) Synthesis and reactions of chloroalkene epoxides. J. Org. Chem. 43, 3596-3600. Lanzaxoae N. A. and McCarty P. L. (1990) Column studies on methanotrophlc degradation of trichloroethene and 1,2-dichloroethane. Grnd Wat. 28, 910-919. Little C. D., Palumbo A. V., Herbes S. E., Lidstrom M. E., Tyndall R. L. and Gilmer P. J. (1988) Trichloroethylene
biodegradation by a methane-oxidizing bacterium. Appl. envir. Microbioi. 54, 951-956. Love O. T. Jr and Eilers R. G. (1982) Treatment of drinking water containing trichloroethylene and related industrial solvents. J. Am. War. Wks Ass. 74, 413--425. Nester E. W., Roberts C. E., Lidstrom M. E., Pearsall N. N. and Nester M. T. (1983) Microbiology, 3rd edition. Saunders, New York. Oldenhuis R., Roedzes J. Y., van der Waarde J. J. and Janssen D. B. (1991) Kinetics of chlorinated hydrocarbon degradation by Methylosinus trichosporium OB3b and toxicity of trichloroethylene. Appl. envir. Microbiol. 57, 7-14. Oldenhuis R., Vink R. L. J. M., Janssen D. B. and Witholt B. (1989) Degradation of chlorinated hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. Appl. envir. Microbiol. 55, 2819--2826. Semprini L., Hopkins G. D., Roberts P. V., Grbic-Galic D. and McCarty P. L. (1991) A field evaluation of in-situ biodegradation of chlorinated ethenes: Part 3, studies of competitive inhibition. Grnd Wat. 29, 239-250. Semprini L., Roberts P. V., Hopkins G. D. and McCarty P. L. (1990) A field evaluation of in-situ biodegradation of chlorinated ethenes: Part 2, results of biostimulation and biotransformation experiments. Grnd War. 28, 715-727. Stanley S. H., Prior S. D., Leak D. J. and Dalton H. (1983) Copper stress underlies the fundamental change in intracellular location of methane mono-oxygenase in methaneoxidizing organisms: studies in batch and continuous cultures. Biotechnol. Lett. 5, 487--492. Strand S. E. and Shlppert L. (1986) Oxidation ofchloroform in an aerobic soil exposed to natural gas. AppL envir. Microbiol. 52, 203-205. Strandberg G. W., Donaldson T. L. and Farr L. L. (1989) Degradation of trichloroethylene and trans-l,2-dichloroethylene by a methanotrophlc consortium in a fixed-film, packed-bed reactor. Envir. Sci. Technol. 23, 1422-1425. Tsien H., Brnsseau G. A., Hanson R. S. and Wackett L. P. (1989) Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b. Appl. envir. Microbiol. 55, 3155-3161. Vogel T. M. and McCarty P. L. (1985) Biotransformation of tetrachloroethylene to trichloroethylene, dichloroethylene, vinyl chloride, and carbon dioxide under methanogenic conditions. Appl. envir. Microbiol. 49, 1080-1083. Vogel T. M., Criddle C. S. and McCarty P. L. (1987) Transformation of halogenated aliphatic compounds. Envir. Sci. Technol. 21, 722-736. Westrick J. J., Mello J. W. and Thomas R. F. (1984) The groundwater supply survey. J. Am. Wat. Wks Ass. 76, 52-59. Whittenbury R., Phillips K. C. and Wilkinson J. F. (1970) Enrichment, isolation and some properties of methaneutilizing bacteria. J. gen. Microbiol. 61, 205-218. Wilson J. T. and Wilson B. H. (1985) Biotransformation of trichloroethylene in soil. Appl. envir. Microbial. 49, 242-243. Windholz M. (1983) The Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals, 10th edition. Merck, Rahway, N.J.
0043-13M/93 $5.00+0.00 Copyright © 1993 Pergamon Press Ltd
DIFFERENT ABILITIES OF EIGHT MIXED CULTURES OF METHANE-OXIDIZING BACTERIA TO D E G R A D E TCE K1M BROHOLM, THOMASH. CHgZS~SEN* and BJORNK. JENSEN Department of Environmental Engineering, Technical University of Denmark, Building 115, DK-2800 Lynghy, Denmark (First received November 1991; accepted in revised form August 1992)
Al~traet--The ability of eight mixed cultures of methane-oxidizing bacteria to degrade trichloroethylene (TCE) was examined in laboratory batch experiments. This is one of the first reported works studying TCE degradation by mixed cultures of methane-oxidizing bacteria at 10°C, a common temperature for soils and groundwaters. Only three of the eight mixed cultures were able to degrade TCE, or to degrade TCE fast enough to result in a significant removal of TCE within the experimental time, when the cultures used methane as growth substrate. The same three mixed cultures were able to degrade TCE when they oxidized methanol, but only for a limited time period of about 5 days. Several explanations for the discontinued degradation of TCE are given. An experiment carried out to re-activate the methane-oxidizing bacteria after 8 days of growth on methanol by adding methane did not immediately result in degradation of methane and TCE. During the first 10-15 days after the addition of methane a significant degradation of methane and a minor degradation of TCE were observed. This experiment revealed that the ability of mixed cultures of methane-oxidizing bacteria to degrade TCE varied significantly even though the cultures were grown under the same conditions. Key words--methane-oxidizing bacteria, chlorinated aliphatics, biodegradation, methane, methanol, trichloroethylene
INTRODUCTION
Chlorinated aliphatics are among the most widespread contaminants in subsurface environments due to their common use in industry for degreasing, dry cleaning and as solvents (Love and Eilers, 1982; Westrick et al., 1984). Research is currently been done to develop new methods for remediation of soil and groundwater contaminated with chlorinated aliphatics. Biological treatment of contaminated soil and groundwater, based on the ability of microorganisms to degrade these contaminants, is a promising method. Chlorinated aliphatics can be degraded under both aerobic and anaerobic (methanogenic) redox conditions. Under methanogeuic redox conditions chlorinated aliphatics are degraded by a reductive dechlorination where, as an example, tetrachloroethylene is degraded to TCE (Vogel et al., 1987). Unfortunately, the anaerobic degradation may result in an accumulation of less chlorinated aliphatic compounds, such as vinyl chloride (Vogel et al., 1987), which is a more hazardous compound than the parent compounds because of its carcinogenic properties (Windholz, 1983). Complete degradation of vinyl chloride to ethylene and carbon dioxide under anaerobic conditions has been observed only in a few studies (Vogel and McCarty, 1985; Freedman and Gossett, 1989). Under aerobic conditions the chlorinated aliphatics can be degraded cometabolically by special *Author to whom all correspondence should be addressed.
groups of bacteria, e.g. methane-oxidizing bacteria (Wilson and Wilson, 1985; Fogei et al., 1986; Strand and Shippert, 1986; Henson et al., 1988, 1989). Generally, the degradation rates increase with decreasing extent of chlorination, and tetrachloroethylene seems to be persistent (Fogel et aL, 1986; Henson et al., 1989). The pathways for aerobic cometabolic degradation of the chlorinated aliphatics have been investigated for TCE by Little et al. (1988). This pathway results in a complete mineralizaton of TCE to carbon dioxide and HCI via TCE-epoxide and other C r and C2-compounds. Some of these TCE degradation products have been proposed to inhibit TCE degradation under certain circumstances (Alvarez-Cohen and McCarty, 1991; Henry and Grbic-Galic, 1991; Oldenhuis et al., 1991). Despite this, aerobic degradation of TCE seems to be one of the most useful processes for remediation purposes. Methane-oxidizing bacteria are widespread in environments where both methane and oxygen are present, such as in transition zones between aerobic and anaerobic areas in soils or sediments (Hanson, 1980). The methane-oxidizing bacteria have been studied extensively during the last 20 years, and the pathway for degradation of methane (see Fig. 1) is well known (Dalton and Stifling, 1982). The first step, which is an oxidation of methane to methanol, is carried out by the enzyme methane monooxygenase, MMO, and requires an electron donor, such as NADH2, which is generated in the later oxidation steps. Methanol is oxidized to formaldehyde, which 215
216
IGM BROHOLM et al. o,
NADH=
H,o
NAD*
X
X~
NAD*
NAD%
NAD"
NAD~
AJ~milmwd into ~luar I ~ m m
Fig. 1. The pathway for methane degradation (Dalton and Stifling, 1982). is either assimilated into bacterial biomass or is further oxidized to formate and carbon dioxide generating N A D H 2 required for the initialoxidation of methane. M M O seems to be responsiblefor the initial oxidation of the chlorinated aliphatics(Fogel et aI., 1986). From the pathway shown in Fig. l it can be seen that methanol may be an alternative growth substrate (electrondonor and carbon source) for the methane-oxidizing bacteria, whereas formate only may serve as an alternativeelectron donor. Laboratory column and fieldexperiments investigating remediation schemes have revealed that it is difficultto achieve an effectivedegradation of T C E (and other chlorinatedaliphatics)in soiland groundwater (Semprini et aI., 1990; Broholm et aI., 1991). Therefore itisimperative to optimize the degradation process in order to develop a feasible remediation scheme. Several chemical factors influence the degradation of chlorinated aliphatics.The copper concentration seems to control whether methane-oxidizing bacteria express soluble or particulate M M O (Stanley et aI., 1983). During copper limitation,when the soluble form is expressed, higher degradation rates for the chlorinated aliphaticshave been observed compared to situationswith copper in excess (Oldcnhuis et aI., 1989; Tsien et aI., 1989). Studies on the interactions between T C E and methane degradation have revealed that the methane concentration has a negative effecton the degradation of T C E because of competition between T C E and methane for M M O (Lanzarone and McCarty, 1990; Broholm et aI., 1992; Oldenhuis et aL, 1991; Semprini et aI., 1991). The highest T C E degradation rateshave been observed by resting cells in the absence of additional methane. Unfortunately, resting cells have only a finiteT C E degradation capacity which may be due to toxicityof some of the T C E degradation products (AlvarczCohen and McCarty, 1991; Oldenhuis et aI., 1991). The use of alternative electron donors such as methanol and formate has been proposed to avoid the competition between T C E and methane. Methanol and formate are both miscible with water in contrast to methane which only has a limited solubility in water of 24 mg/l at 20°C 0Vindholz, 1983). Formate has been used successfully as an electron donor to enhance the T C E degradation by resting cellsfor shorter periods of time (from a few
hours to a few days) (Oldenhuis et al., 1989; AlvarezCohen and McCarty, 1991; Semprini et al., 1991), whereas the use of methanol so far has resulted in differing results (Janssen et al., 1988; Oldenhuis et al., 1989). Henry and Grbic-Galic (1990) have shown that the TCE degradation rates for different mixed and pure cultures of methane-oxidizing bacteria grown in slightly different mineral media varied by more than a factor of 10. Besides the above-mentioned chemical factors affecting the degradation of chlorinated aliphatics, the nature of the actual microbial consortia may also be an important factor. If highly efficient cultures could be identified they might be used as inoculum for rvmediation purposes. The reported experiments on TCE degradation involving mixed and pure cultures of methane-oxidizing bacteria apparently reveal large variations in TCE degradation rates, but so far only one investigation of the diversity among mixed cultures of methane-oxidizing bacteria to degrade TCE has been reported (Henry and Grbic-Galic, 1991). That investigation revealed large differences between TCE degradation rates for resting cells of three mixed cultures. Generally, the reported experiments have been carried out at temperatures between 20 and 35°C, which are much higher than the natural groundwater temperature. Therefore, the degradation of chlorinated afiphatics by methane-oxidizing bacteria has to be studied at groundwater temperatures in order to make the obtained results more applicable for natural groundwater and soil systems. This paper reports the results of laboratory experiments carried out at 10°C investigating the ability of eight mixed cultures of methane-oxidizing bacteria to degrade TCE. Mixed cultures, in contrast to pure cultures, are investigated since an initially amended pure culture is likely to develop into a mixed culture in non-sterile environments, such as soils and groundwaters, being supplied with methane and oxygen. Furthermore, it is more difficult to obtain pure cultures than mixed cultures. The inocula for the mixed cultures were collected at waterworks that treat methane-containing groundwater for public water supply. The aeration and filtration units of such waterworks contain relatively stable transition zones between a reduced, methane-rich environment (anaerobic groundwater) and an aerobic environment,
TCE degradation by mixed cultures of methane-oxidizing bacteria creating ideal conditions for m e t h a n e - o x i d i z i n g bacteria. This study covers three experimental series. T h e first series of experiments focused o n t h e ability o f the eight mixed cultures to degrade T C E d u r i n g m e t h a n e oxidation, while the second series o f experiments investigated the ability of the same m i x e d cultures to degrade T C E during methanol oxidation. T h e third experimental series investigated the effect o f alternating methane a n d methanol additions u s i n g m e t h a n e for enzyme induction and m e t h a n o l for bacterial growth. MATERIALS AND METHODS
The mixed cultures of methane-oxidizing bacteria Eight mixed cultures of methane-oxidizing bacteria were obtained from seven different Danish waterworks that treat groundwater containing methane. Material taken from the sand and/or the aeration filter was used as inoculum. The inocuia for the Cultures 1, 2, 3 and 5A originated from sand filters consisting of sand or gravel, while the inocula for the Cultures 4, 5C, 6 and 7 originated from aeration filter sludge. About 50 mi of each inoculum was mixed with 150 ml of nutrient solution in 1 liter glass bottles. 20 ml of methane was added to the headspace resulting in an initial methane concentration of 2.5% (vol/vol) in headspace and 0.75 mg/l in liquid phase. The bottles were incubated in the dark at 10°C with magnetic stirring. Weekly, about 50 ml of each culture was diluted with 150 ml of nutrient solution and new methane was added. Initially, some of the inocula consisted of solids, but after subsequent dilutions the cultures only consisted of liquid. After about 2 months significant methane consumption and bacterial growth was observed, and the cultures were then used for the batch test experiments. The nutrient solution contained the following macroand micronutrients per liter distilled water: 5.8rag of Na2HPO4"2H20, 3.8mg of NH4C1, 147rag of CaCl 2, 122mg of NaNO 3, 100rag of NaHCO3, 9.4rag of KC1, 0.4mg of FeCI3"6H20, 50rag of MgSO4"7H20, 10/~g of MnCI2"4H20, 12/~g of ZnSO4.7H20, 6/~g of CuSO4" 5H20, 6/zg of CoCI 2.6H20 , 5.5 #g of Na2MoO 4.2H20, 2/~g of Na2B4OT.10H20. The pH of the solution was adjusted to 7. The concentration of some of the individual ions is shown in Table 1. Water samples for characterization of the original environments of the cultures were taken from the same place as the inocula except for Cultures 4 and 5C, where samples
217
were taken from the sand filters. Temperature, O2-concentration and pH were measured on site, whereas the other parameters were determined in the laboratory on water samples preserved in accordance with the analysis. Table 1 shows the chemical characteristics of the water samples representing the original environment of the studied cultures. The chemical composition of the water samples was quite similar except for nitrate, ammonium, sulfate, magnesium and iron, where a difference of more than a factor of ten between the water samples was observed. Also for methane a difference of a factor of ten was observed, probably because Samples 6 and 7 were taken at the aeration filters where the groundwater still contains high concentration of methane due to a yet incomplete stripping of methane. The chemical composition of the nutrient solution was similar to the water samples except that the nutrient solution contains high concentrations of nitrate and phosphate, two essential nutrients for the bacteria and low iron concentration.
Description of the batch experiments All the experiments were carried out in l l 7 m l glass bottles equipped with Miniert valves (DYNATECH Precision Sampling Corporation, Baton Rouge, La), which enabled frequent sampling from the bottles. Each bottle contained 10 ml inoculum and 10 ml nutrient solution. The inocula in all the experiments were mixed cultures of bacteria grown with methane as the sole energy and carbon source. The control bottles contained 20 ml nutrient solution and 0.3 g sodium azide (NAN3) per liter to prevent any bacterial growth. The bottles were incubated in a rotating box (10 rpm) in the dark at 10°C, the groundwater temperature in Denmark (see Table 1). In the TCE degradation experiment with methane as growth substrate the batches contained TCE at initial liquid concentrations of 500 pg/l and 0.17 mmol methane. Methane partitioned between the liquid phase and the headspace resulting in an initial concentration of methane in the liquid phase of 0.075 retool/1 (1.2 mg/l). Methane was added to the headspace with a syringe. 50/~1 of a stock solution of TCE, prepared by dissolving a known volume of TCE in a nutrient solution, was added to each batch. In the TCE degradation experiment with methanol as growth substrate, the batches contained TCE at initial liquid concentrations of 500 ~g/l and 0.17 mmol methanol. The initial methanol concentration in the liquid phase was 8.5mmol/l (272mg/1). Methanol was dissolved in the nutrient solution before it was added. In the experimental series with alternating methanol and methane addition the initial liquid concentration of TCE was 500 ~g/i and the initial concentration of methanol in the liquid phase 8.5 retool/1. In this experiment both methane
Table 1. Chemical characterization of the water samples taken at the waterworks and the nutrient solution. The water samples from Waterworks 1, 2, 3, 4 and 5 were taken from the sand filter and 6 and 7 were from the aeration filter Nutrient Waterworks 1 2 3 4 5 6 7 solution Temperature (°C) 9.3 11.0 9.6 11.4 10.0 10.2 NA NA pH 7.27 7.78 7.23 8.16 7.32 7.77 7.76 7.0 Oxygen (mg/I) 9.9 10.0 9.0 9.3 9.5 9.4 10.1 NA Methane (rag/I) 0.23 0.98 0.02 0.87 0.04 1.08 4.00 0 TAL (mequlv/I)* 5.5 5.8 5.4 7.9 4.5 5.4 6.9 1.0~ TOC (rag C/I)t 2.1 3.9 2.6 5.7 1.3 4.2 4.9 0 Nitrate (rag N/I) 0.02 0.04 0.16 0.15 0.13 0.07 0.01 20 Ammonium (mg N/I) 0.70 0.83 0.18 !.8 0.17 0.56 1.2 1.0 Sulfate (rag/I) 13 7 63 13 52 i2 8 20 Phosphate (rag/l) 0.19 0.22 0.07 0.14 0.06 0.09 0.26 3. I Calcium (rag/I) 99 91 127 ! 15 116 122 98 57 Magnesium (rag/I) 18 22 20 66 17 18 23 4.9 Iron (mg/I) 2.2 2.3 1.4 0.3 1.2 1.2 1.8 0.09 Manganese (mg/l) 0.06 0.06 0.08 <0.02 0.06 <0.02 0.08 0.003 Copper ~g/I) 1.4 0.8 1.0 5.0 0.8 0.6 1.2 1.5 *TAL = total alkalinity; tTOC = total organic carbon; :~NAffinot analyzed; ~calculated.
218
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219
TCE degradation by mixed cultures of methane-oxidizing bacteria
in the control experiments within 30 days was about 10% of the initial amount of TCE. The molar concentration of methane shown in Figs 2 and 4 is calculated as if all the methane at the time of sampling was in the liquid phase.
initial biomass concentration in water samples at the start of the experiments was measured as protein. The relative TCE concentration shown in Figs 2 and 3 is calculated as (C/Co)/(C~u~/C~u~.o) where C and Co=u~ are liquid TCE concentrations at the time of sampling in the degradation batches and in the control batches, Co and C~o~,o are initial 5quld TCE concentrations in the degradation batches and in the control batches. The loss of TCE
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Fig. 4. The degradation of methanol and methane for one mixed culture (Culture 7) of methane-oxidizing bacteria and a control in an experiment where the bacteria grew only on methanol the first 8 days. At day 8 methane and methanol were added to the batches, and further methanol was added at day 15. The individual results of three batches and the average (dashed lines) are shown in the figure. The molar concentration of methane was calculated as if all methane at the sampling time was in the liquid phase. Protein measurements were performed on fiquid samples to which 3 M trichloroacetic acid solution was added to kill the bacteria before storing at 4°C. The samples were centrifuged for 20rain at 4000 rpm in a Heraens Christ Labofuge III. The supernatant was discarded carefully and 0.66 N sodium hydroxide solution was added to the pellet Analytical procedures to lyse the bacteria. After incubation for 2 days at 35°C and staining with Coomamie brilliant blue G-250 the protein Methane, methanol and TCE were analyzed on a DANI 8520 gas chromatograph equipped with a flame ionization content was measured spectropbotometrically at 595 um by detector for methane and methanol and an electron capture a Bechman DB GT grating spectrophotometer. The preparation of Coomassie brilliant blue G-250 and the protein detector for TCE. Chromatograph separation was obtained using a 30 m long penetrated open tubular fused silica standards are described by Bradford (1976). pH was measured with a Microprocessor pH Meter column (0.53 mm i.d.) coated with CP-SIL5 cross bounded. pH 196, WTW, and oxygen and temperature were measured The gas chromatograph was operated isothermally at a with a Microprocessor Oximeter oxi 196, WTW. column temperature of 50°C for methane and TCE, and Calcium, magnesium, manganese and iron were analyzed I05°C for methanol, and detector temperature of 275°C for both detectors. The carrier gas was nitrogen. Data acqui- by means of flame atomic absorption spectrophotometry on sition and integrations were performed on a MAXIMA a Perkin-Elmer 370. Copper was measured on an inductively coupled plasma mass spectrometer (IPC-MS) Chromatography Workstation. Air samples for methane were injected with a gas-tight (Perkin-Elmer 5000). Total organic carbon (TOC) was syringe and the concentrations were determined by compari- measured on a Dohrmann DC 180. Nitrate, ammonium, sulphate and phosphate were anason with a standard curve. Methane in water was measured on samples taken with a syringe and injected into a vial with lyzed on a Technicon AutoAnalyser. Nitrate was detera vacuum. After methane had equilibrated between the mined by use of a hydraziue reduction method based on a water and the headepace, the concentration of methane in modification of a procedure of Kamphake et al. 0967). the headspace was measured as previously described and the P-mmonium WaS determined by use of a phenate method original methane concentration in the water sample was described in APHA (1989), sulfate by use of an automated methylthymol blue method described in ALPHA (1989) and calculated. TCE was measured on air samples which were injected phosphate by use of an ascorbic acid method described in directly and the concentrations were determined by com- APHA (1989). Total alkalinity (TAL) was measured on a Radiometer parison with standards made in pentane. The concentration of TCE in the liquid phase was calculated from the Autoburette ABU 11 equipped with a Radiometer Research measured concentration in the headspace tufing a dimension- pH Meter PHM 64. The rumples were titrated with H2SO4 less Henry's law constant of 0.17 at 10°C (Gossett, 1987). to pH 4.5.
sodium azide, >99%, and TCE, >99.5%, were purchased from Merck, Darmstadt, Germany. Coomassie brilliant blue G-250 and bovine albumin for protein standards were purchased from Sigma Chemical Co., St Louis, Mo., U.S.A. The nutrients were of reagent grade quality.
TCE degradation by mixed cultures of methane-oxidizing bacteria RESULTS
The mixed cultures used as inocula in all the experiments used methane as the sole energy and carbon source. They are referred to as mixed cultures of methane-oxidizing bacteria even though they consist of methane-oxidizing bacteria and other bacteria. Most methane-oxidizing bacteria are capable of using methanol as growth substrate as well as methane (Whittenbury et al., 1970). The mixed cultures probably contained methane-oxidizing bacteria (have MMO, are capable of using methane and methanol as growth substrate and may be able to degrade TCE), methanol-oxidizing bacteria (do not have MMO, are able to use methanol as growth substrate and probably not able to degrade TCE) and other bacteria.
Degradation of TCE with methane as growth substrate Significant TCE degradation was observed for only three of the eight mixed cultures (Cultures 4, 5A and 7), when methane was added as growth substrate. The TCE and methane degradation curves for these three mixed cultures and for Culture 3, which was unable to degrade TCE, are shown in Fig. 2. At the end of the experiments after 30 days the average observed degradation of TCE was between 28 and 55% of the initial amount of TCE for the three mixed cultures (Cultures 4, 5A and 7) (Fig. 2). The fact that no TCE degradation was observed within 30 days for the other five mixed cultures (Cultures 1, 2, 3, 5C and 6) does not necessarily mean that these cultures are unable to degrade TCE. The TCE degradation rates for the bacteria in these mixed cultures may be so low that they would not result in significant removal of TCE within the experimental time. The results of the TCE degradation experiments with methane as growth substrate are summarized in Table 2, where the time period for 50% degradation of the initial amount of methane for all eight mixed cultures is shown together with the ability of the mixed cultures to degrade TCE. A mixed culture was considered to degrade TCE if more than 10% of the initial amount of TCE was removed within the experimental time. The time period until 50% of the initial amount of methane was degraded varied between 7 and 17 days for the eight mixed cultures (Table 2). All the cultures except Culture 5C de-
221
graded more than 90% of the initially added methane within 30 days. The three mixed cultures which degraded a significant amount o f TCE were not the fastest methane degrading cultures. The initial concentration of biomass in each experiment measured as protein is also shown in Table 2. The initial concentration o f protein in the experiments was between 2.5 and 14.4mg]l [equal to 10~-6 × 10~ bacteria/ml, using conversion factors of 0.5mg protein/rag cell dry weight (Nester et al., 1983), and 2 x 109bacteria]mg cell dry weight (Balkwill et al., 1988)]. The protein concentration is not a specific measurement of the number of methane-oxidizing bacteria in the mixed cultures, but only gives the total amount of bacteria in the mixed cultures. It is obvious that the methane-oxidizing bacteria in the mixed cultures are responsible for the observed degradation of methane and TCE.
Degradation of TCE with methanol as growth substrate The results of the TCE degradation experiments for four of the eight mixed cultures (Cultures 3, 4, 5A and 7) of bacteria with methanol as growth substrate are shown in Fig. 3. The mixed cultures used as inoculum in these experiments grew with methane as the only carbon and energy source before the start of the experiment. An average TCE degradation of 22-40% of the initial amount of TCE was observed for three mixed cultures (Cultures 4, 5A and 7) (Fig. 3). The mixed cultures for which a significant degradation of TCE was observed when methanol was added as growth substrate were the same mixed cultures for which a significant TCE degradation was observed when methane was added as growth substrate. The degradation of TCE stopped after about 5 days even though the mixed cultures continued to degrade methanol. At the time when the bacteria had degraded all the initially added methanol, the batches were re-fed with methanol (data not shown in Fig. 3). The results of the TCE degradation experiments with methanol as growth substrate are summarized in Table 2, where the time period for 50% degradation of the initial amount of methanol for all eight mixed cultures is shown together with the ability of the mixed cultures to degrade TCE when the
Table 2. Summaryof the TCE degradationexperimentsfor 8 mixed culturesof methane-oxidizingbacteriawith methane or methanol as growth substrate. The time period in days for 50% degradationof the growth substrateis shown together with the mixedculturesabifities for degradingTCE with methane and methanol.Degradationof TCE was consideredsignificantif more than 10"/, of TCE was removed. The initial protein concentrationin the experimentsis also shown Culture number 1" 2* 3* 4t 5A* 5Ct 6t 7t Initial protein cone. (rag/I) 2.5 3.6 14.4 10.2 Time period for 50% degradationof methane (days) 12 9 7 11 TCE degradation with methane + Time period for 50% degradationof methanol (days) 6 4 2 3 TCE degradationwith methanol + *The mixed culture originated from sand filtersat a groundwater-lmsedwaterworks. tThe mixed culture orisinated from aeration filtersat a groundwater-basedwaterworks.
9.3 12 + 4 +
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222
Kn~ BROHOLMet al.
mixed cultures used methanol as growth substrate. Methanol degradation was observed for all eight mixed cultures (Table 2). The time period for 50% degradation of the initial amount of methanol varied between 2 and 6 days (Table 2). The initial concentration of biomass in each experiment measured as protein was the same as in the degradation experiments where methane was added as growth substrate (Table 2). It is impossible to distinguish between the methanol degradation by methane-oxidizing bacteria and the methanol degradation by methanol-oxidizing bacteria based on the methods used in this study. Alternating methane and methanol addition
These experiments were carried out in a similar way as the experiments with only methanol as growth substrate except that methane was added to the batches at day 8. Before the experiment (day 0) the mixed cultures (Cultures 4, 5A and 7) had only grown with methane as the sole growth substrate. From day 0 to day 8 the bacteria only grew with methanol, whereas both methane and methanol were present from day 8 and to the end of the experiments. TCE was added to the experiments at day 0. The removal of methane and methanol for Culture 7 and a control experiment is shown in Fig. 4. The results for Cultures 4 and 5A are similar to the results for Culture 7. A fast methanol degradation was observed during the whole experiment. At the time methane was added at day 8 and until day 19 only a minor methane and no TCE degradation was observed. After day 19 a significant methane and a minor TCE degradation was observed. The reason why methane was added at day 8 was that the experiment with methanol as growth substrate revealed that the mixed cultures did not retain the ability to degrade TCE while using methanol as growth substrate. If the methane-oxidizing bacteria lost the ability to degrade TCE because they did not produce MMO while oxidizing methanol, then the addition of methane at day 8 should result in a production of MMO followed by a degradation of TCE. DISCUSSION
Eight mixed cultures were obtained from different inocula, but were grown at the same growth conditions: at 10°C, with the same nutrient solution, and with methane as the sole carbon and energy source. TCE degradation experiments with methane as growth substrate showed that only three of the eight mixed cultures were able to degrade TCE or able to degrade TCE fast enough to result in a significant TCE degradation within 30 days. The three mixed cultures (Cultures 4, 5A and 7) which were able to degrade TCE were not the fastest methane-degrading cultures (Table 2). As the mixed cultures were grown at similar conditions the observed differences with
respect to TCE degradation between the methaneoxidizing bacteria in the mixed cultures are probably due to different inherent abilities of the methaneoxidizing bacteria to degrade TCE. The same three mixed cultures, which were able to degrade TCE when the mixed cultures used methane as growth substrate, were able to degrade TCE when the cultures used methanol as growth substrate. Unfortunately, the TCE degradation occurred only within the first 5 days even though methanol was still present in the batches at that time. Obviously, methanol-degrading bacteria were present in the batches during the whole experiments, whereas methane-oxidizing bacteria was present at the start of the experiments and may not have been active for more than 5 days. This indicates that it was the methane-oxidizing bacteria in the mixed cultures which were responsible for the observed degradation of TCE in the experiments with methanol as growth substrate. At least three explanations why the methane-oxidizing bacteria did not continue to degrade TCE with methanol as growth substrate instead of methane are given below. One explanation is that the degradation of TCE results in production of TCE-epoxide or other intermediate products, which may be toxic to the methane-oxidizing bacteria. The experiments, reported in the literature, showing inhibition of resting cells of methane-oxidizing bacteria by TCE degradation products have been carried out with relatively high TCE concentration and with relatively high biomass to achieve a fast degradation of TCE and a fast inactivation of the cells (few hours) (Alvarez-Cohen and McCarty, 1991; Oldenhuis et al., 1991). In contrast to this, our experiments were carried out with relatively low TCE concentration and relatively low biomass resulting in a slow TCE degradation and a slow inactivation of the cells (5 days). Our observations have been confirmed at field conditions by Semprini et al. (1991) who successfully used methanol as alternative substrate to support TCE degradation for 60 h. The field experiment was carried out with low biomass concentration and low TCE concentration, but the time period where the bacteria used methanol was not sufficiently long to show whether inactivation of the cells occurred. Degradation studies with c/s- and trans-l,2-dichloroethylene (c1,2-DCE and t-I,2-DCE) by mixed cultures of methane-oxidizing bacteria in the presence of methane have revealed accumulation of c- and t-l,2DCE-epoxide outside the cells, which shows that c-I,2-DCE and t-I,2-DCE degradation products may not bind to the cells at these growth conditions (.Ianssen et al., 1988; Strandberg et al., 1989). This is not necessarily valid also for TCE. The half-lives for chemical conversion of TCE-epoxide of 1.3min (Kline et al., 1978), for c-l,2-DCE-epoxide of about 72h and for t-l,2-DCE-epoxide of about 30h (Janssen et al., 1988), make it more likely to observe accumulation of the epoxide of c- or t-I,2-DCE than
TCE degradation by mixed cultures of methane-oxidizing bacteria of TCE. This indicates that the TCE degradation products may be accessible for further conversion, and for this reason, the time period until total inactivation of the cells becomes an important factor. In our experiments with relatively slow TCE degradation the TCE degradation products may have sufficient time to undergo further conversion, which makes an inhibition of the cells by the degradation products more unlikely than in experiments with a fast TCE degradation. However, any final conclusion whether or not the observed inactivation of the cells is due to inhibitory effect by TCE degradation products cannot be made. A second explanation is that the methane-oxidizing bacteria in the mixed cultures do not get the required energy (NADH2) for the initial oxidation of TCE. When methane-oxidizing bacteria oxidize methanol they produce N A D H 2 which theoretically could supply MMO with the required energy for TCE oxidation. Therefore it is unlikely that the discontinued TCE degradation was due to lack of energy. The third explanation is that the methane-oxidizing bacteria in the mixed cultures do not produce MMO in absence of methane. The observed TCE degradation in our experiments which stopped after 5 days may be due to some MMO the methane-oxidizing bacteria already had produced when the bacteria grew on methane before the start of the experiments. The literature contains contradictory results about the induction of MMO. A pure culture of methaneoxidizing bacteria (Methylosinus trichosporium OB3b) grown on methanol has been shown to retain the activity of MMO for 9 months (Best and Higgins, 1981). Therefore, it is possible for some methaneoxidizing bacteria to continue degrading TCE using methanol as growth substrate, if the TCE degradation products do not inhibit the bacteria. However, the methane-oxidizing bacteria in the mixed cultures in our experiments do not seem to retain their MMO activity in absence of methane assuming that the bacteria were not inhibited by TCE degradation products or did not lack energy for the continuing degradation of TCE. The result of the experiment with alternating methane and methanol addition showed no immediate methane degradation at the time methane was added after 8 days where the bacteria oxidized methanol (Fig. 4). It took about 10-15 days before a significant methane degradation and a minor TCE degradation was observed. The fact that no immediate methane degradation was observed at day 8 showed that either the methane-oxidizing bacteria were not present at that time or were not re-activated immediately. After 10-15 days methane degradation was observed revealing that methane-oxidizing bacteria were also present at day 8 but the number of methane-oxidizing bacteria may be too small to result in a significant methane degradation. No degradation of T e E was observed for Cultures 4, 5A and 7 within the first 5 days of this experiment,
223
which is in contrast to previous experiments. AlvarezCohen and McCarty (1991) have shown that the amount of TCE degraded by resting cells depends on several factors, e.g. the time period the cells have been in absence of methane. This may explain why the same cultures in one case degrade a certain amount of TCE for 5 days and in another case do not degrade a significant amount of TCE. The fact that no or very little TCE degradation occurred within the first 8 days of this experiment eliminates the explanation that the methane-oxidizing bacteria at day 8 were inactivated by TCE degradation products. CONCLUSION Eight mixed cultures of methane-oxidizing bacteria grown in similar conditions showed different abilities to degrade TCE when using either methane or methanol as growth substrate. For only three mixed cultures (Cultures 4, 5A and 7) was a significant TCE degradation observed within the experimental time when the mixed cultures used methane as growth substrate. When the same three mixed cultures were oxidizing methanol they were able to degrade TCE, but the ability was present only during the first 5 days of the experiments. It was not possible to re-activate the methane-oxidizing bacteria after 8 days of methanol oxidation by adding methane. The observation that some of the mixed cultures did not degrade TCE (or degraded TCE very slowly) and some degraded TCE relatively fast under similar growth conditions is an important observation dealing with bioremediation of soil or groundwater. If an indigenous population of methane-oxidizing bacteria is present in the soil and/or groundwater, and stimulated by the addition of methane and oxygen, then the required time period for /n situ bioremediation depends on what kind of methane-oxidizing bacteria are present at the actual site. In order to inoculate soils and/or groundwaters or on-site treatment plants with methane-oxidizing bacteria it is important to inoculate with a culture, which is able to degrade TCE (or other chlorinated aliphatics) fast at the actual growth conditions. Acknowledgements--The financial support from the National Agency of Environmental Protection in Denmark is gratefully acknowledged. We thank Michael I.~fvall, Department of Environmental Engineering, Technical University of Denmark, for help with the experiments and the analysis. REFERIgNCI~
Aivarez-Cohen L. and McCarty P. L. (1991) TCE transformation by a mixed methanotrophic cuiture-effects of toxicity, aeration and reductant supply. Appl. envir. Microbioi. 57, 228-235. APHA (1989) Standard Methods for the Examination of Water and Wastewater, 17th edition. American Public Health Association, Washington, D.C. Balkwill D. L., Leach F. R., Wilson J. T., McNabb J. F. and White D. C. (1988) Equivalence of microbial biomass
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measures based on membrane lipid and cell wall components, adenosine triphosphate, and direct counts in subsurface aquifer sediments. Microb. Ecol. 16, 73-84. Best D..I. and I-Iiggins I. J. (1981) Methane-oxidizing activity and membrane morphology in a methanol-grown obligate methanotroph Methylosinus trichosporium OB3b. J. gen. Microbiol. 125, 73-84. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Analyt. Biochem. 72, 248-254. Brobolm K., Christensen T. H. and Jonson B. K. (1991) Laboratory feasibility studies on biological in-situ treatment of a sandy soil contaminated with chlorinated aliphatics. Envir. TeclmoL 12, 279-289. Broholm K., Christensen T. H. and Jonson B. K. (1992) Modelling TCE degradation by a mixed culture of methane-oxidizing bacteria. Wat. Res. 26, 1177-I 185. Dalton H. and Stirring D. I. (1982) Co-metabolism. Phil. Trans. R. Soc. Lond. 297, 481-496. Fogel M. M., Taddeo A. R. and Fogel S. (1986) Biodegradation of chlorinated ethenes by a methane-utilizing mixed culture. Appl. envir. Microbiol. 51, 720-724. Freedman D. L. and Gossett J. M. (1989) Biological reductive dechlorination of tetrachloroethylene and trichloroethylene to ethylene under methanogenic conditions. Appl. envir. Microbiol. 55, 2144-2151. Gossett J. M. (1987) Measurement of Henry's law constant for C, and C2 chlorinated hydrocarbons. Envir. Sci. Technol. 21, 202-208. Hanson R. S. (1980) Ecology and diversity of methylotrophlc organisms. Adv. appl. Microbiol. 26, 3--39. Henry S. M. and Grbic-Galic D. (1990) Effect of mineral media on trichloroethylene oxidation by aquifer methanotrophs. Microb. Ecol. 20, 151-169. Henry S. M. and Grbic-Galic D. (1991) Influence of endogenous and exogenous electron donors and trichloroethylene oxidation toxicity on trichloroethylene oxidation by methanotrophlc cultures from a groundwater aquifer. Appl. envir. Microbiol. 57, 236-244. Henson J. M., Yates M. V. and Cochran J. W. (1989) Metabolism of chlorinated methanes, ethanes, and ethylenes by a mixed bacterial culture growing on methane. J. Ind. Microbiol. 4, 29-36. Henson J. M., Yates M. V., Cochran J. W. and Shackleford D. L. (1988) Microbial removal of halogenated methanes, ethanes, and ethylenes in an aerobic soil exposed to methane. FEMS Microbiol. EcoL 53, 193-201. Janssen D. B., Grobben G., Hoektra R., Oldenhuis R. and Witholt B. (1988) Degradation of trans-l,2-dichloroethene by mixed and pure cultures of methanotrophic bacteria. Appl. Microbiol. Biotechnol. 54, 951-956. Kampbake L. J., Hannah S. A. and Cohen J. M. (1967) Automated analysis for nitrate by hydrazine reduction. War. Resour. Res. 1, 205-216. Kline S. A., Solomon J. J. and Duuren B. L. V. (1978) Synthesis and reactions of chloroalkene epoxides. J. Org. Chem. 43, 3596-3600. Lanzaxoae N. A. and McCarty P. L. (1990) Column studies on methanotrophlc degradation of trichloroethene and 1,2-dichloroethane. Grnd Wat. 28, 910-919. Little C. D., Palumbo A. V., Herbes S. E., Lidstrom M. E., Tyndall R. L. and Gilmer P. J. (1988) Trichloroethylene
biodegradation by a methane-oxidizing bacterium. Appl. envir. Microbioi. 54, 951-956. Love O. T. Jr and Eilers R. G. (1982) Treatment of drinking water containing trichloroethylene and related industrial solvents. J. Am. War. Wks Ass. 74, 413--425. Nester E. W., Roberts C. E., Lidstrom M. E., Pearsall N. N. and Nester M. T. (1983) Microbiology, 3rd edition. Saunders, New York. Oldenhuis R., Roedzes J. Y., van der Waarde J. J. and Janssen D. B. (1991) Kinetics of chlorinated hydrocarbon degradation by Methylosinus trichosporium OB3b and toxicity of trichloroethylene. Appl. envir. Microbiol. 57, 7-14. Oldenhuis R., Vink R. L. J. M., Janssen D. B. and Witholt B. (1989) Degradation of chlorinated hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. Appl. envir. Microbiol. 55, 2819--2826. Semprini L., Hopkins G. D., Roberts P. V., Grbic-Galic D. and McCarty P. L. (1991) A field evaluation of in-situ biodegradation of chlorinated ethenes: Part 3, studies of competitive inhibition. Grnd Wat. 29, 239-250. Semprini L., Roberts P. V., Hopkins G. D. and McCarty P. L. (1990) A field evaluation of in-situ biodegradation of chlorinated ethenes: Part 2, results of biostimulation and biotransformation experiments. Grnd War. 28, 715-727. Stanley S. H., Prior S. D., Leak D. J. and Dalton H. (1983) Copper stress underlies the fundamental change in intracellular location of methane mono-oxygenase in methaneoxidizing organisms: studies in batch and continuous cultures. Biotechnol. Lett. 5, 487--492. Strand S. E. and Shlppert L. (1986) Oxidation ofchloroform in an aerobic soil exposed to natural gas. AppL envir. Microbiol. 52, 203-205. Strandberg G. W., Donaldson T. L. and Farr L. L. (1989) Degradation of trichloroethylene and trans-l,2-dichloroethylene by a methanotrophlc consortium in a fixed-film, packed-bed reactor. Envir. Sci. Technol. 23, 1422-1425. Tsien H., Brnsseau G. A., Hanson R. S. and Wackett L. P. (1989) Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b. Appl. envir. Microbiol. 55, 3155-3161. Vogel T. M. and McCarty P. L. (1985) Biotransformation of tetrachloroethylene to trichloroethylene, dichloroethylene, vinyl chloride, and carbon dioxide under methanogenic conditions. Appl. envir. Microbiol. 49, 1080-1083. Vogel T. M., Criddle C. S. and McCarty P. L. (1987) Transformation of halogenated aliphatic compounds. Envir. Sci. Technol. 21, 722-736. Westrick J. J., Mello J. W. and Thomas R. F. (1984) The groundwater supply survey. J. Am. Wat. Wks Ass. 76, 52-59. Whittenbury R., Phillips K. C. and Wilkinson J. F. (1970) Enrichment, isolation and some properties of methaneutilizing bacteria. J. gen. Microbiol. 61, 205-218. Wilson J. T. and Wilson B. H. (1985) Biotransformation of trichloroethylene in soil. Appl. envir. Microbial. 49, 242-243. Windholz M. (1983) The Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals, 10th edition. Merck, Rahway, N.J.