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Journal of Environmental Quality 31:144-149 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORT
Bioremediation and Biodegradation

Impact of Methylene Chloride on Microorganisms and Phenanthrene Mineralization in Soil

Egbert Schwartz*, Sinh V. Trinh and Kate M. Scow

Graduate Group in Ecology and Dep. of Land, Air and Water Resources, One Shields Ave., Univ. of California at Davis, Davis, CA 95616-8627

* Corresponding author (eschwart{at}du.edu)

Received for publication January 29, 2001.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study investigated the effects of the quantity of methylene chloride, used as a carrier solvent for phenanthrene when added to soil, on phenanthrene mineralization kinetics, soil phospholipid fatty acid profiles (PLFA), and phenanthrene distribution. Methylene chloride dosages of 25 µL/g soil or more resulted in an enrichment of saturated PLFAs, suggesting soil microorganisms had adjusted their cell membranes in response to the solvent. A greater fraction of phenanthrene mineralized when spiked in 5 µL/g than in 25 µL/g methylene chloride suggesting that the methylene chloride became toxic to phenanthrene-degrading organisms in soil. Phenanthrene was more equally distributed among 0.1 g soil subsamples if spiked in 25 than 5 or 1 µL methylene chloride per gram soil. Thus the amount of methylene chloride used to spike phenanthrene in soil strongly impacted the mineralization kinetics, phenanthrene distribution, and microbial community in soil. Because a variety of spiking methods are used in biodegradation research, scientists should consider the quantity of solvents used when comparing results among different studies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
WATER INSOLUBLE POLLUTANTS that sorb strongly in soils are presently a high research priority because these compounds often remain recalcitrant in the environment (Alexander, 1995). Polycyclic aromatic hydocarbons (PAHs) are examples of such pollutants and are toxic or potential carcinogens. Therefore a large body of work on the degradation of PAHs in soil has appeared in the literature in the last couple decades (Pignatello and Xing, 1996; Scow and Johnson, 1997). Often these studies concern the degradation of freshly added pollutants to soil. Because PAHs have low water solubility they must first be dissolved in a solvent before being spiked into soil. Given that environmentally relevant concentrations of pollutants are often very low, solvents are typically added to soil in greater quantities than the PAHs. The effects of the quantity and identity of solvent on the microbial community and PAH degradation kinetics have not been widely investigated even though solvents can potentially have a big impact on either. Solvents can be used as carbon sources for microbial growth, they can be toxic to biodegrading populations, and their physical properties such as how miscible they are with water may influence the distribution of PAHs in soil (Alexander, 1995; Pignatello and Xing, 1996).

Methylene chloride is commonly used as a solvent to deliver phenanthrene, a PAH, to soil because it can be metabolized by only a small group of organisms. It is very volatile and thus leaves soil rapidly after the PAHs are spiked into soil (Verschueren, 1983; Montgomery and Welkom, 1990). Methylene chloride can be toxic to bacteria (Kanazawa and Filip, 1986; Byers and Sly, 1993). It is not miscible with water and therefore does not readily penetrate the soil solution (Verschueren, 1983; Montgomery and Welkom, 1990). Because we have used methylene chloride extensively in our studies on phenanthrene sorption and biodegradation in soil (Scow et al., 1986; Scow et al., 1994; Johnson and Scow, 1999; Scow and Johnson, 1997; Schwartz and Scow, 1999; Schwartz et al., 2000) we investigated the effect of methylene chloride on phenanthrene degradation and the microbial community. Here we report our findings on the effect of methylene chloride dosage on soil PLFA patterns, phenanthrene distribution and phenanthrene mineralization kinetics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Spatial Distribution of Carbon-14-Phenanthrene in Soil
Twenty g of sterile Yolo soil were amended with 100 ng/g 14C-phenanthrene (Sigma Chemical Co., >98% purity, specific activity of 59 mCi/mmol) in either 20, 100, or 500 µL of methylene chloride. Yolo soil is a fine silty mixed, non-acid, thermic, Typic Xerothent, further described in (Scow et al., 1994). It was collected from the top 15 cm of a tomato field at the student farm, University of California, Davis. The soil was passed through a 2 mm sieve, and stored at 4°C. One week before use the soil moisture was brought up to -0.222 bars, or 15.5% with deionized water. The phenanthrene was labeled on the inner ring at the C-9 position. Soil was sterilized by {gamma}-irradiation with a Cobalt-60 irradiator at 355299 rad/hr for 14.083 h (Nuclear reactor laboratory, the University of Michigan). Phenanthrene was mixed into the soil with a spatula by hand for 2 min.

Thirty subsamples of 0.1(±0.01) g of soil were immediately removed from each of 3 replicate microcosms. One ml of methanol was added to each subsample after placement in 7 ml scintillation vials and the sample was shaken for one hour. Five ml of scintillation cocktail was added and the radioactivity of each sample was measured in a liquid scintillation counter (Beckman Instruments, Fullerton, CA). The amount of radioactivity in a 0.1 g subsample was normalized by dividing it by the mean radioactivity of all samples spiked with a similar volume of methylene chloride. The normalized values were subsequently plotted in a rank abundance graph.

Mineralization of Phenanthrene in Yolo Soil
Either 25 ng/g or 100 ng/g phenanthrene per g soil was added in 20, 100, or 500 µL of methylene chloride to 20 g of soil. Phenanthrene used in all experiments was a mixture of 0.045 µCi of 14C-labeled phenanthrene (Sigma Chemical Co., >98% purity, specific activity of 59 mCi/mmol) with non-isotopically labeled phenanthrene. Phenanthrene was mixed into 20 grams of soil with a spatula by hand for 2 min. The soil was incubated in an airtight pint (approximately 480 ml) mason jar (Alltrista Corp., Muncie, IN). The jar contained a vial with 1 ml of 0.5 N NaOH to trap CO2 evolving from soil. Three replicate samples were prepared for each experimental treatment. The base was periodically sampled and its radioactivity was measured in a liquid scintillation counter (Beckman Instruments, Fullerton, CA). The mason jar was opened during sampling to ensure sufficient oxygen remained available over the course of the incubation.

Phospholipid Fatty Acid Analysis
Effects of methylene chloride on the microbial community were investigated by adding 100 ng/g phenanthrene to 20 g of soil in 20, 100, 500, and 2500 µL of methylene chloride. Phospholipid fatty acid extractions were performed on 8 g of Yolo soil, as described previously (Bossio and Scow, 1998; White et al., 1997) on soils immediately before, 24 h after, and 360 h after, phenanthrene was spiked into soil. A correspondence analysis was performed on phospholipid fatty acids results with SAS software (SAS Institute, Cary, NC). A Tukey-Kramer analysis was performed with JMP software (SAS Institute, Cary, NC) to determine if significant differences occurred among the treatments in ratios of saturated to unsaturated fatty acids and the quantities of the fatty acids 14:0 iso and 15:0 anteiso.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Variation in the amount of radioactivity among 0.1 g subsamples was greater in soils spiked with phenanthrene in 1 µL/g than 5 or 25 µL/g methylene chloride (Fig. 1) . One subsample contained 69% more radioactivity than the mean radioactivity content among all subsamples taken from soil spiked with 1 µL/g of methylene chloride. The smallest variance was observed among subsamples spiked with phenanthrene in 25 µL/g of solvent. The subsample with greatest radioactivity among these samples only contained 18% more radioactivity than the mean. Mineralization of 25 ng/g or 100 ng/g phenanthrene was more rapid if the phenanthrene was delivered in 1 or 5 µL/g than in 25 µL/g methylene chloride (Fig. 2) . Phenanthrene mineralization rates reached a maximum 66 h after addition for soils amended with 1 or 5 µL/g methylene chloride and 162 h after the phenanthrene amendment for the 25 µL/g spike. Though more phenanthrene was mineralized in soils to which phenanthrene was added in 1 or 5 µL/g, after 210 h the difference between cumulative amounts of phenanthrene mineralized in all soils became smaller. The variance in phenanthrene mineralization rates was greater in soils to which phenanthrene was added in 25 than 1 or 5 µL/g methylene chloride.



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  		Fig. 1. Rank abundance diagram of radioactivity in 0.1-gram soil subsamples. Twenty grams dry weight soil was spiked with 100 ng/g phenanthrene in either 20 µL ({circ}), 100 µL ({square}), or 500 µL ({Delta}) methylene chloride.

 


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  		Fig. 2. Degradation of 25 ng/g (A) and 100 ng/g (B) phenanthrene spiked in 20 µL ({blacktriangleup}), 100 µL ({blacksquare}), or 500 µL (•) methylene chloride. Error bars represent two standard deviations.

 
Effects of 25 and 125 µL/g methylene chloride on soil microorganisms, as measured by PLFA analysis, were apparent within 24 h after phenanthrene addition. Soils into which phenanthrene was spiked with 1 or 5 µL/g methylene chloride had similar PLFA patterns to soils to which no methylene chloride was added (Fig. 3) . In contrast, PLFA patterns of soils spiked with phenanthrene in 25 or 125 µL/g methylene chloride, were very different from those of soils that were not exposed to phenanthrene or methylene chloride (Fig. 3). The first two axes of the correspondence analysis plot explained 86% of the variation within the PLFA data set. PLFA profiles of soils, 15 d after phenanthrene was added, were different from patterns of soils 1 d after phenanthrene was added (Fig. 3). Fifteen days after the phenanthrene was added to soil between 22 and 36% of the phenanthrene had mineralized (Fig. 2). Separation between samples taken on days 1 and 15 by correspondence analysis was not as pronounced as the separation caused by methylene chloride dosage (Fig. 3).



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  		Fig. 3. Correspondence analysis of PLFA patterns of soils spiked with 100 ng/g phenanthrene; c = day 0. The first part of the number describes the volume of methylene chloride used to add phenanthrene to 20 g of soil. The end of the number refers to the number of days after phenanthrene addition that the soil was sampled (i.e., 201 = 20 µL of methylene chloride sampled one day after phenanthrene addition).

 
There was no significant difference between the molar ratios of saturated to unsaturated fatty acids of soils to which either no phenanthrene or methylene chloride was added, or soils spiked with phenanthrene in 1 or 5 µL/g methylene chloride (Fig. 4) . In contrast, PLFA patterns from soils that were spiked with phenanthrene in 25 or 125 µL/g methylene chloride were enriched in unsaturated fatty acids. The molar ratios of saturated to saturated fatty acids of the 25 µL/g and 125 µL/g methylene chloride treatments increased 23 and 41% over the molar ratio of the 5 µL/g methylene chloride treatment respectively. The separation of PLFA patterns from different dosage samples in the correspondence analysis plot were largely due to the saturated fatty acids (Fig. 5) . In particular, the quantity of the fatty acids 14:0 iso and 15:0 anteiso increased 97 and 31% in samples to which phenanthrene was added in 25 or 125 µL/g methylene chloride respectively as compared with soils spiked with 5 µL/g methylene chloride (Fig. 6) .



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  		Fig. 4. Molar ratios of saturated to unsaturated fatty acids in soils spiked with 100 ng/g phenanthrene. All samples, except the before sample, were taken one day after the phenanthrene was added to soil. Numbers refer to the quantity, in µL per g of soil, of methylene chloride used to deliver phenanthrene. Error bars equal two standard deviations. Significant differences ({alpha} = 0.05) only exist between treatments labeled with different letters.

 


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  		Fig. 5. Loadings of fatty acids in correspondence analysis of PLFA pattern from soils exposed to different doses of methylene chloride. The loadings describe the impact of individual values (i.e., PLFAs) on the separation between the treatments (i.e., methylene chloride dosage and sample time). Correspondence analysis of samples is shown in Figure 3.

 


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  		Fig. 6. Nano moles of the fatty acids 14:0 iso (top) and 15:0 anteiso (bottom) extracted from soil. C= soil before experiment was started, 0 = soil to which neither phenanthrene nor methylene chloride was added, 1, 5, 25, and 125 = soils to which 100 ng/g phenanthrene was added in 1, 5, 25 or 125 µL/g methylene chloride, respectively. Significant differences ({alpha} = 0.05) only exist between treatments labeled with different letters.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The spatial heterogeneity of phenanthrene in soil, as indicated by variation in phenanthrene concentrations of 0.1 g soil subsamples (Fig. 1), was higher in microcosms spiked with small volumes of methylene chloride. Reid et al., 1998, also found that spiking procedure impacted the distribution of poorly water-soluble compounds in soil. Degradation kinetics are probably dominated by processes occurring at smaller scales than the approximately 0.1 cm3 of soil we studied (Luthy et al., 1997). The variance in phenanthrene concentrations could be even greater at the smaller spatial scales.

Degradation of phenanthrene may proceed faster if the compound is distributed completely homogeneously in soil. In our study, a slightly greater fraction of phenanthrene spiked in 5 µL/g of methylene chloride degraded than phenanthrene added to soil in 1 µL/g of methylene chloride (Fig. 2). In studies with phenanthrene concentrations in the ppb range, such as ours, sites capable of supporting growth of phenanthrene-degrading bacteria will be present only if phenanthrene is unevenly distributed. In microsites with concentrations of phenanthrene sufficient for growth there will be a lag in degradation because population growth requires time (Simkins and Alexander, 1984). This lag will effect how much phenanthrene eventually will be degraded because phenanthrene sorbs rapidly in soil, thereby becoming increasingly less bioavailable (Schwartz and Scow, 1999).

The concentration of phenanthrene will also influence the fraction that sorbs (Scow et al., 1994; Pignatello and Xing, 1996). Sorption is often described by a Freundlich isotherm (Chen et al., 1999; Schultz et al., 1999). In cases where this nonlinear isotherm is applicable, a positive relationship exists between the fraction of pollutant in the aqueous phase and the total pollutant concentration in soil. Thus, in microsites with locally high pollutant concentrations a larger percentage of phenanthrene is aqueous and therefore available for degradation.

Spiking phenanthrene in 25 µL/g, in comparison to 1 and 5 µL/g, of methylene chloride resulted in lower phenanthrene mineralization rates (Fig. 2), suggesting the solvent was toxic to phenanthrene-degrading populations. The toxicity of high doses of methylene chloride is reflected in the PLFA analysis. The PLFA profiles of microbial communities did not change until at least 25 or 125 µL/g methylene chloride was added to the soil. Moreover, saturated fatty acids, such as iso 14:0 and anteiso 15:0, were enriched in membranes of soil microbial communities spiked with 25 or 125 µL/g methylene chloride (Fig. 6). Bacteria alter their cell envelope to affect membrane fluidity in the presence of high concentrations of solvents (Keweloh and Heipieper, 1996). They may exchange unsaturated in favor of saturated fatty acids or shift from cis to trans configured double bonds in their PLFA's. Determination of cell envelope changes after exposure to o-xylene showed a solvent-tolerant Pseudomonas putida strain was enriched in saturated fatty acids (Pinkart et al., 1996). A solvent-sensitive strain did not show these changes, suggesting saturated fatty acids aided survival of the solvent-tolerant strain in media containing high organic solvent concentrations. Sublethal concentrations of toluene in a culture of Pseudomonas putida, S12, resulted in an increase of trans-unsaturated fatty acids and in the degree of saturation of membrane fatty acids (Heipieper and Bont, 1994). In the presence of high concentrations of toluene, three Pseudomonas putida strains accumulated trans-unsaturated fatty acids but not saturated fatty acids (Weber et al., 1994). Increases in ratios of saturated to unsaturated fatty acids, unlike shifts in cis to trans type fatty acids, can only be accomplished through de novo synthesis and, thus, require time and energy (Heipieper and Bont, 1994). The lower phenanthrene mineralization rate observed in soils spiked with 25 µL/g methylene chloride (Fig. 2) may reflect the time required for phenanthrene-degrading bacteria to change their ratios of saturated to unsaturated fatty acids. The fatty acids 14:0 iso and 15:0 anteiso which were especially enriched in soil bacterial communities exposed to 25 or 125 µL/g methylene chloride are not signature or biomarker lipids (White et al., 1997) and thus can not be used to identify the appearance of a new population.

Toxic effects of methylene chloride on cultures of bacteria have been demonstrated at concentrations below those used in this study. Kanazawa and Filip, 1986, observed that 100 µg of methylene chloride in 100 g of soil inhibited the activity of ß-glucosidase, ß-acetylglucosaminidase, and proteinase for 28 d, after which time the enzymatic activity of the soil recovered to control soil levels. Activity of all enzymes used in the study declined after 1000 µg of methylene chloride was added to 100 g of soil, but again the soil recovered its full potential for catalysis within 2 mo. Application of 100 µL of methylene chloride to 5 g of air-dried soil delayed oxidation of NH4 but had no impact on mineralization of glucose or naphthalene. Oxidation of NH4 recovered completely after 27 d. Growth of five obligate methanotrophic bacteria of the genera, Methylomonas, Methylosinus, and Methylocystis ceased if approximately 650 mg/L methylene chloride was included in the broth (Byers and Sly, 1993) and addition of 6500 mg/L methylene chloride was bactericidal. In comparison, we added 26.52, 132.6, 663, 3315 mg of methylene chloride to 3.1 ml of soil solution, resulting in final concentrations of 8.50, 41.44, 184.17, and 591.96 g/L, respectively. With the exception of the 8.50 g/L concentration, these concentrations are above the water solubility limit which is approximately 13 g/L (Verschueren, 1983; Montgomery and Welkom, 1990). Most of the methylene chloride spiked into soil, however, evaporated, due to its low vapor pressure and high Henry's law coefficient (2.68 x 10-3 atm m3/mole) (Verschueren, 1983; Montgomery and Welkom, 1990), immediately after it was added to soil. As a result, we do not know the concentrations of methylene chloride to which the soil microbial community was exposed. Therefore we don't know if the PLFA profiles and the mineralization kinetics were affected by methylene chloride in the aqueous phase or by vapors in the incubation jars.

The high variations observed in degradation kinetics of replicates spiked with 500 µL of methylene chloride may be due to disparity in recovery or adaptation rates of microbial populations between microcosm replicates. For instance, phenanthrene mineralization in the 3rd replicate microcosm of soils spiked with 25 ng/g phenanthrene in 25 µL/g methylene chloride was consistently higher than in the other replicates. After 505 h, 32.7% of phenanthrene had mineralized in the 3rd microcosm while only 24.1 and 18.8% had mineralized in the 1st and 2nd microcosms respectively. The variance in mineralization rates was therefore not due to errors in measurements but in actual differences in mineralization rates between replicate microcosms. The 3rd microcosm appeared to rebound more rapidly from the toxic effects of the methylene chloride. Recovery from toxic exposure may have involved growth in which case small initial differences resulted in large variations further on in the experiment because growth is an exponential process. Once methylene chloride was volatilized from the soil there were good opportunities for growth because populations affected by the methylene chloride may have abandoned niches that could be newly exploited. The higher variance in the 25 µL/g treatment thus were consistent with the observation that high methylene chloride doses were toxic to the microbial community.

The data presented in this paper does not suggest that methylene chloride was used as a carbon or energy source. For instance, we did not observe an increase in the quantity of PLFAs extracted from soils with larger doses of methylene chloride. Though methylene chloride can be biodegraded in a variety of soils, most studies report degradation of much lower concentrations of the solvent than we used in our study (Davis and Madsen, 1991; Ergas et al., 1994).

The quantity of methylene chloride used to deliver phenanthrene to soil impacts degradation kinetics in at least two independent ways. Use of large quantities of the solvent will promote a homogeneous distribution of the phenanthrene in soil, which will limit complications due to large variances in phenanthrene concentrations in soil microsites. However, too much methylene chloride will have toxic effects on the soil microbial community and the degradation kinetics will reflect death and subsequent recovery of phenanthrene-degrading microorganisms. Both processes are difficult to model and are usually ignored in studies of phenanthrene degradation in soil. Thus, in choosing how much solvent to use to add phenanthrene to soil, a compromise must be made between toxicity and homogenous distribution. Based on our studies, using 5 µL/g methylene chloride to add phenanthrene to soil appears a reasonable compromise.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





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