Methane oxidation and microbial exopolymer production in
land®ll cover soil
Helene A. Hilger
a,*, David F. Cranford
a, Morton A. Barlaz
ba
Department of Civil Engineering, University of North Carolina at Charlotte, Charlotte, NC 28223, USA b
Department of Civil Engineering, North Carolina State University, Raleigh, NC 27695-7908, USA
Accepted 30 May 1999
Abstract
In laboratory simulations of methane oxidation in land®ll cover soil, methane consumption consistently increased to a peak value and then declined to a lower steady-state value. It was hypothesized that a gradual accumulation of exopolymeric substances (EPS) contributed to decreased methane uptake by clogging soil pores or limiting gas diusion. This study was conducted to detect and quantify EPS in soil from columns sparged with synthetic land®ll gas and from fresh land®ll cover cores. Polysaccharide accumulations were detected with alcian blue stain. EPS was observed adhering to soil particles and as strands associated with, but separate from soil grains. Glucose concentrations in laboratory soil columns averaged 426 mg kgÿ1 dry soil, while in a column sparged with air the average glucose concentration in a horizon was 3.2 mg glucose kgÿ1 dry soil. Average glucose concentrations in two of four cores sampled from a closed land®ll ranged from 600±1100 mg kgÿ1 dry soil, while control cores averaged 38 mg glucose kgÿ1dry soil. Viscosity due to EPS was measured by comparing ®ltration rates of soil suspensions. Soil extracts from the upper horizons of laboratory columns sparged with land®ll gas ®ltered at about one-third the rate of extracts from the lower horizons, and the land®ll core with the highest glucose content also produced highly viscous extracts. Breakthrough curves measured in columns before and after methane exposure were similar, so that short-circuiting due to clogging was not occurring. The data support the hypothesis that EPS impeded oxygen diusion to an active bio®lm and limited the extent of methane oxidation.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Land®ll; Methanotrophs; Methane oxidation; Exopolymer; Polysaccharides
1. Introduction
Methane migration through land®ll caps is the fourth largest source of anthropogenic CH4 emissions
worldwide (Stern and Kaufmann, 1996) and it is the largest source in the United States (US Department of Energy, 1997). These emissions alter the global CH4
budget, and since CH4is a potent greenhouse gas, they
contribute to global climate change.
Microbial CH4 consumption in the aerobic portions
of a land®ll cap reduces CH4 emissions to the
atmos-phere, and the degree to which this occurs and the
conditions that promote it are all under investigation (Whalen et al., 1990; Jones and Nedwell, 1993; Kjeld-sen et al., 1997). Laboratory and ®eld studies indicate that CH4 oxidizers typically consume 10±20% of the
CH4 passing through a land®ll cover, although under
laboratory conditions, up to 60% CH4 oxidation has
been reported (Kightley et al., 1995). Bogner et al. (1995) have shown that under certain conditions, land-®ll covers are even a sink for atmospheric CH4.
Some of the factors that in¯uence microbial CH4
oxidation in land®lls include climate variables such as moisture and temperature (Jones and Nedwell, 1993; Bogner et al., 1995; Czepiel et al., 1995; Boeckx and Van Cleemput, 1996; Borjesson and Svensson, 1997), as well as CH4 concentration (Czepiel et al., 1996; Soil Biology & Biochemistry 32 (2000) 457±467
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* Corresponding author. Fax: +1-704-510-6953.
Bogner et al., 1997), soil type (Kightley et al., 1995) and pH (Hilger et al., 2000).
In long term (80±120 d) laboratory simulations of CH4 oxidation in land®ll cover soil, CH4 uptake has
exhibited a peak followed by a decrease to a lower steady-state value (Hoeks, 1972; Kightley et al., 1995; Hilger et al., 2000). Potential explanations for this decline include the production of inhibitory substances, protozoan grazing, nutrient depletion, or an accumu-lation of extracellular polymers that either clogs soil pores and causes short-circuiting or impedes gas diu-sion into the cells.
Many bacteria, including CH4 oxidizers, produce
exopolymeric substances (EPS) that can serve as a source of anchorage. EPS production may also oer resistance to desiccation, a shield from predators, and a mechanism to keep certain populations in close proximity (Fletcher et al., 1992). The nature and degree of polymer formation vary widely amongst both microbial species and environmental conditions, and EPS production has been linked to both nutrient imbalance and O2de®ciency (Wrangstadh et al., 1986).
EPS accumulation can alter the metabolism of bac-teria embedded in a bio®lm. Composed largely of polysaccharide (Costerton et al., 1981), a viscous ®lm can oer greater resistance to substrate diusing into the base ®lm (Christensen et al., 1990; Mozes et al., 1992) and there is evidence that diusivity decreases with increasing ®lm age (Matson and Characklis, 1976).
Methanotrophs are known to produce EPS both as capsules (Wyss and Moreland, 1968; Whittenbury et al., 1970) and as copious slime (Hou et al., 1978; Jen-sen et al., 1991). Chida et al. (1983) described two polymers produced by a single thermophilic methano-troph. The polymers had molecular weights of 120,000±340,000, sugar contents ranging from 37± 56%, and amino acid contents between 30 and 38%. Southgate and Goodwin (1989) reported both viscous and non-viscous EPS production in pure cultures of
Methylophilus methylotrophus, and the polysaccharides contained sugars as well as acetate and pyruvate resi-dues. A highly viscous polymer produced by
Methylo-philus viscogenes is harvested and marketed under the
name of Poly 54 (Leak et al., 1992). It has been suggested that for methanotrophs in particular, pro-duction of a carbon-rich polymer is used as a meta-bolic mechanism to prevent formaldehyde accumulation when carbon is in excess (Linton et al., 1986).
Methanotroph bio®lms have been studied and exploited for a variety of degradation processes (Bilbo et al., 1992; Fennell et al., 1992; Bowman et al., 1993; Sly et al., 1993; Arcangeli and Arvin, 1997), but there has been little study of methanotrophs and EPS pro-duction in the soil vadose zone. Our objectives were to
(1) evaluate whether CH4 oxidation in land®ll cover
soil promoted EPS production, (2) examine the nature and quantity of EPS produced during CH4 oxidation
and (3) evaluate whether this EPS could cause short circuiting of gas fed to soil columns.
2. Materials and methods
2.1. Experimental design
A series of experiments was conducted to investigate whether signi®cant EPS production occurs in soil exposed to land®ll gas. Tests to measure the presence of EPS were conducted on soils removed from labora-tory columns that had been gassed with CH4 for
sev-eral thousand hours, on fresh soil cores collected from a land®ll cover, and on soil cores from sites with no history of CH4exposure.
EPS was detected qualitatively by staining samples with alcian blue. This cationic stain is used to detect polysaccharide with light microscopy (Kiernan, 1990; Fassel et al., 1992). EPS polysaccharides contain a var-iety of anionic moieties (Costerton et al., 1981; Van Iterson, 1984) and the stain is believed to bind to these by forming electrostatic or ionic linkages (Scott et al., 1964; Scott, 1972).
Quantitative tests included measurement of the ®l-terability of soil suspensions and assays of ®ltered soil extracts for glucose. Total carbon (%C) and relative CH4 oxidation potential were also measured on
selected samples.
To evaluate the importance of soil-pore clogging on CH4 gas short-circuiting, breakthrough curves were
measured in soil columns at start up and after 2400± 3300 h of gassing with a 50/50 CH4/CO2 synthetic
land®ll gas mixture. A reduced retention time due to short-circuiting would decrease CH4uptake.
2.2. Soil description
The soil used to ®ll the columns was a sandy loam collected from the cover of a closed land®ll with a his-tory of CH4production. Fresh ®eld core samples were
collected from the Renaissance Park land®ll (Char-lotte, NC), which is closed and has been converted to recreational ®elds. Control cores with exposure to only atmospheric CH4(1.7ml lÿ1) were taken from the
Uni-versity of North Carolina at Charlotte campus.
2.3. Soil columns
Four laboratory soil columns were used to measure CH4breakthrough curves and to provide soil for EPS
tests. Soil column reactors were constructed from 15 cm dia PVC pipe and contained a 30 cm column of soil (Fig. 1). A drain in the bottom of the pipe, over-lain by 15 cm of sterile gravel, supported the soil. After packing, perforated stainless steel needle probes (3.2-mm dia) were inserted through gas-tight ports in the column wall into the compacted soil. The probes penetrated to the middle of the column cross-section. Needle ports were capped on the outside with a fabri-cated brass ®tting that permitted a double septa barrier between the soil and ambient air. An additional sampling port was placed at the top of the headspace over the soil. The columns were capped gas-tight except for an exit port at the top, which vented out-doors. After capping, the headspace volume was ap-proximately 5.3 l.
2.4. Column ®lling
Soil columns were prepared within 2 d of soil collec-tion. The soil moisture content was adjusted to 152
0.5% before ®lling a column. Soil was packed in six layers of equal mass. After each layer was added, a 4.5 kg standard Proctor compaction hammer (ASTM D 1557-78 in Liu and Evett, 1996) was used to deliver 10 evenly distributed blows over the horizon surface area. The hammer drop distance per blow was based on pre-liminary tests to calibrate the compaction to various
hydraulic conductivities (K). Columns were prepared with a hydraulic conductivity of 10ÿ5cm sÿ1.
2.5. Column operation and monitoring
A synthetic land®ll gas (LFG) containing a 50/50 mix of CO2/CH4 was delivered at 3.2510
ÿ7
g CH4
cmÿ2 sÿ1 (10 cm3 LFG minÿ1) through a port at the base of the column. A 50-cm3 minÿ1 ¯ow of air entered near the soil surface, so that the only source of O2 in the column was that which diused vertically
from the top. Routine monitoring included measure-ment of inlet air and land®ll gas ¯ow rates and the exit gas ¯ow rate and composition.
Columns were dismantled as needed for soil tests. Columns 1, 2 and 3 were dismantled at 2424, 2808 and 4128 h, respectively. Column 4 was not dismantled during this series of experiments and, therefore, data related to soil after gassing do not include measures of soil in column 4.
2.6. Gas analysis
Gas ¯ow measurements were performed with a J&W Scienti®c ¯owmeter (Model ADM-2000 Folsom, CA), and gas concentrations were measured by gas chroma-tography (GC). 50 ml gas samples were analyzed on a Shimadzu 14a GC (Columbia, MD) equipped with a CTR1 column (Alltech, Deer®eld, IL) and a thermal conductivity detector. The carrier gas was He at 60 cm3minÿ1. Injector and oven temperatures were main-tained at 658C, and the detector temperature was 758C. Standard curves were generated using external standards each time the GC was used. A mass balance was performed on each gas using ¯ow and gas concen-tration measurements.
2.7. Breakthrough curves
Initial breakthrough curves were obtained by with-drawing headspace gas samples hourly for 22 h after initiation of LFG and air ¯ows into the columns. Final breakthrough curves were obtained after 2400± 3300 h of LFG exposure, once it was apparent that CH4 uptake had peaked and established a steady state
consumption level. First, the inlet LFG was replaced by 100±150 cm3 minÿ1 of N2 ¯ow to sparge O2 and
CH4 from the column. LFG was then reinitiated at 10
cm3 minÿ1, but a 50 cm3 minÿ1 N2 ¯ow was used in
place of air at the top of the column so that O2
con-sumption would not confound the results. Hourly headspace gas monitoring was repeated for 24±30 h, after which normal air¯ow was re-established.
Fig. 1. Soil column reactor design.
2.8. Column sampling
At the termination of a soil column trial, soil was collected from each of six 5-cm horizons, with the top designated horizon 1 and the bottom horizon 6. The ®eld cores were sliced from the top down into 5-cm horizons. Soil was tested for EPS by staining, ®lterabil-ity, glucose and total carbon analyses. The relative CH4 oxidation potential by horizon was measured in
two soil columns.
2.9. Polysaccharide staining
Soil samples from each horizon of the laboratory columns were diluted 1:100 in pH 7 phosphate buer (0.3 g KH2PO4, 0.7 g K2HPO4), after which 3±4 drops
containing soil particles were placed on a slide and topped with a cover slip. A 1% alcian blue solution in ethanol was diluted to 0.1% with deionized water and used to stain polysaccharide present on the soil par-ticles. Several drops of stain were placed on the slide and wicked across to cover the soil. After 3 min, the stain was rinsed several times by repeated wicking of phosphate buer. The slides were observed with a Bausch and Lomb Balplan microscope (Rochester, NY).
2.10. Filterability rate
Soil was mechanically extracted in 1 M KCl (1 part soil: 5 parts solution) for 1 h. 15 cm3 of well-mixed slurry were then quickly poured into a porcelain cruci-ble that was lined with Whatman No. 1 ®lter paper. The rate of gravity ®ltration of the slurry was measured over 1 h.
2.11. Glucose assays
A portion of the unused soil slurry prepared for ®l-terability tests was centrifuged and vacuum ®ltered through a GF-C glass ®ber ®lter. The ®ltrate was tested for saccharides using a modi®cation of the Dubois colorimetric test (Dubois et al., 1956) described by Deng and Tabatabai (1994). Glucose standards were prepared in 1 M KCl.
2.12. Total carbon
Total carbon was measured on 20 mg air dried, sieved samples with a Perkin Elmer 2400 CHN El-emental Analyser.
2.13. Methane oxidation potential
The measurement of CH4 oxidation potential has
been described and is summarized here (Hilger et al.,
2000). 8-g soil samples adjusted to 1520.5% moisture content were sealed in 45 cm3 vials. 7 cm3 of head-space air were removed and replaced with an equal volume of 50/50 CH4/CO2. Headspace CH4 depletion
was monitored over 2±3 d and compared to sterile soil controls.
3. Results
3.1. Soil columns
At peak consumption, the soil removed 45±50% of the input CH4, and this decreased to 15±20% at steady
state (Fig. 2). Pro®les of average gas concentrations at steady state and ®nal soil pH by horizon are shown in Table 1. The columns remained oxygenated through-out, and a distinct pH gradient developed. Fresh soil pH was 6.3, but soil removed from the top of the reac-tors had a pH of 5.2 and the pH increased steadily to 6.3 in the bottom soil horizon.
Initial and ®nal CH4 breakthrough curves were
quite similar, although there was a slight trend toward
Fig. 2. Methane consumption in four replicate soil columns sparged with synthetic land®ll gas.
faster CH4migration after gassing (Fig. 3). If excessive
EPS production was occurring over time, then it was not causing pronounced short-circuiting of gas ¯ow through the reactors.
3.2. Polysaccharide staining
Soil sampled from the laboratory columns and stained with alcian blue indicated that there was more EPS in the upper horizons. Slides from this region could clearly be distinguished by clumped soil particles bound and thickly coated with dense blue-stained EPS (Fig. 4a). Wide strands of blue-stained material linked some of the clumps or ¯oated separately (Fig. 4b), suggesting that they had sucient rheological stability to withstand disturbances imposed during slide prep-aration.
Soil sampled from the lower horizons showed less blue-stained EPS, with only portions of a soil grain outlined in blue or two relatively unstained grains held together by blue-stained polysaccharide. Soil from lower horizons appeared as numerous small particles uniformly dispersed with much less particle aggrega-tion than upper horizon samples.
Dierences between both the thickness of polymer and the tendency of soil grains to aggregate in the upper and lower horizons may re¯ect the relative quantities of biomass present, dierences in the nature of the polymers in each region, or both. Fassel et al. (1992) studied methanotroph EPS using a variety of staining techniques and reported that Methylosinus
tri-chosporiumOB3b, a type II methanotroph, had both a
dense inner layer of exopolymer and a ®brous outer layer. Although it is not presumed that the alcian blue stain results here correspond directly with the EPS forms observed by Fassel, et al., their report does sub-stantiate the potential for a physical distinction between two forms of polymer in the same methano-troph population.
3.3. Filterability
Filterability tests provided relative measures of the viscosity of soil extracts. Tests were performed on fresh soil used to ®ll the columns, soil from col-umns after gassing, soil from colcol-umns sparged with air instead of CH4, fresh land®ll cover core samples
and fresh cores from soil with exposure to atmospheric CH4 only. Small ®ltrate volumes represent high
vis-cosity and presumably high EPS (Table 2). Soil from the upper three horizons of columns gassed with CH4
was dicult to ®lter, while there was little resistance to ®ltration in soil from horizons 5 and 6. Fresh land®ll soil used for ®lling the columns also ®ltered readily (2.3 cm3hÿ1).
The ®lterability measures of all column or core horizons within a treatment were averaged for stat-istical comparisons. Where laboratory columns and ®eld cores were compared, only horizons from the top 20 cm of the columns were considered in order to be consistent with the 20 cm depth of the cores. The ®lterability of ®eld control cores with no LFG exposure were so similar to each other that only the averages for each horizon are shown in Table 2. A comparison of the averages between treatments showed that soil from laboratory columns gassed with LFG was signi®cantly more resistant to ®ltration than soil from a column gassed with air P<0:001 or soil
from the four control sites P<0:001). Three of the
four land®ll cores (1,2 and 4) ®ltered as readily as con-trol cores. Land®ll core 3 was much more resistant
P<0:001 than the controls but not signi®cantly
dierent from the land®ll-gassed laboratory column soil P0:05), con®rming that the polymer
accumu-lation found in the laboratory columns was not an ex-perimental artifact and could be documented in the ®eld as well.
Table 1
Gas concentration pro®les, pH, percent total carbon and C-to-N ratios in replicate soil columns sparged with synthetic land®ll gas
Horizon CO2 O2 N2 CH4 pHa Total carbonab Carbon-to-nitrogen ratioa
percent (v vÿ1)c mass percent
1 16.1 13.0 60.3 10.7 5.220.08 0.6620.04 2220.52
2 22.4 9.9 53.2 14.5 5.220.03 0.6520.03 2220.56
3 28.2 7.2 46.2 18.3 5.420.04 0.6020.02 2120.27
4 33.8 5.0 38.7 22.6 6.020.01 0.4320.03 1420.27
5 38.2 3.1 32.4 26.3 6.220.05 0.3420.01 1320.51
6 40.5 2.3 28.3 28.9 6.320.05 0.3220.03 1320.82
a
Value is the average of three replicate reactors with standard error of the mean (SE). Values from a fourth replicate are not included because it had not been dismantled at the time these data were reported.
b
Total percent carbon in fresh soil (0.27) has been subtracted from values shown in the table.
c
Value shown is the average of gas concentrations in one port above and one port below the horizon. Each port value is the average of four soil columns at 2040 h when reactors were at steady state with respect to methane oxidation.
3.4. Glucose in soil extracts
Glucose concentrations were used to quantify the relative amount of bio®lm present in each column or core horizon. As with ®lterability, measures of all col-umn or core horizons within a treatment were aver-aged, and only horizons from the top 20 cm of the laboratory columns were considered in comparisons with ®eld core samples. There were large dierences in glucose concentrations between soils with or without exposure to land®ll gas (Tables 2 and 3). The glucose concentrations in the six horizons of the laboratory column gassed with air only ranged from 0±7.6 mg kgÿ1 dry soil, so that the mean glucose concentration for this treatment was signi®cantly lower P<0:001
than that of soil from laboratory columns gassed with LFG. The mean glucose concentration in the labora-tory columns gassed with LFG was also signi®cantly greater P<0:001 than the mean glucose
concen-tration in the ®eld core samples from control sites. There were large dierences in glucose concentration among the land®ll core samples (Table 3). If, as in the laboratory reactors, elevated glucose concentrations
are associated with CH4 exposure, then dierences
may re¯ect relative amounts of CH4exposure between
sampling sites. This could be due to preferential ¯ow paths for the land®ll gas through a heterogeneous soil. Three of the four land®ll ®eld cores (2±4, Table 3) had average glucose concentrations that were signi®cantly higher P<0:05 than those in the control cores. The
average glucose concentration in the laboratory col-umn sparged with air only was not signi®cantly dier-ent P0:05 from the glucose content of the control
®eld cores.
The Dubois test is commonly used with glucose col-orimetric standards to quantify EPS (Characklis et al., 1990). Since ®lm polymers are a mixture of a number of sugars, it should be noted that if two distinct poly-mers are present in equal mass, but contain dierent amounts of glucose, then the soil with more glucose would appear to contain more EPS.
3.5. Total carbon
Total %C in the fresh soil composite used to form the laboratory soil columns was 0.27%. Total %C
Table 2
Comparison of ®lterability of soil with or without exposure to land®ll gasa
Horizon Depth Soil column+LFGb Soil column +airc Land®ll core 1 Land®ll core 2 Land®ll core 3 Land®ll core 4 Average of 4 control coresd
(cm) ml ®ltrate collected hÿ1kgÿ1dry soil
1 3 0.3620.07 1.9220.01 1.08 1.01 0.35 0.90 1.1920.11
2 7 0.3020.05 1.6520.00 1.11 0.61 0.20 1.48 1.0120.10
3 11 0.2220.04 1.8220.00 1.61 1.18 0.13 1.66 1.1020.08
4 17 0.3520.04 1.8820.01 1.71 1.48 0.49 1.77 1.0720.11
5 22 0.8520.06 1.7920.06
6 27 1.0420.19 1.7920.08
Average glucose all horizons (mg kgÿ1dry soil)e 426 3.2 38 655 1158 108 38
a
No statistics are shown where measures represent one sample per horizon.
b
Values are the average of duplicates from each horizon of columns 1, 2 and 3 (Fig. 1), sampled after 2400, 2800 and 4100 h, respectively,2S.E.
c
Values are the average of duplicate samples from each horizon of a single reactor sampled after 2800 h,2S.E.
dControl cores had exposure to atmospheric methane only. eSee Table 3 for values by horizon.
Table 3
Comparison of glucose content in soil with or without exposure to land®ll gasa
Horizon Depth Soil column+LFG-1b Soil column+LFG-2b Soil column+LFG-3b Land®ll core 1 Land®ll core 2 Land®ll core 3 Land®ll core 4 Range of control coresc
(cm) mg glucose kgÿ1dry soil
1 3 678222 67520.0 59227.0 30 648 746 281 0±92
2 7 685287 72120.0 623221 73 1123 1608 74 12±85
3 11 57821.8 588218 37820.2 23 299 1176 58 0±106
4 17 30720.0 21828.7 192211 26 550 1101 18 0±81
5 22 183240 142216 14428.0
6 27 240225 9621.5 246212
a
No statistics are shown where measures represent one sample per horizon.
b
Columns 1, 2 and 3 are replicates and were sampled after 2400, 2800 and 4100 h, respectively. Values are the average of duplicates from each horizon2S.E.
c
Range of four cores from soil not exposed to land®ll gas.
H.A.
Hilger
et
al.
/
Soil
Biology
&
Biochemistr
y
32
(2000)
457±467
(after correcting for fresh soil carbon) and the carbon-to-nitrogen (C-to-N) ratio in each horizon of soil after gassing with LFG are presented in Table 1. The car-bon accumulation after exposure to LFG was at least double that of the fresh soil. The %C measures from the top three horizons of all columns (n = 12) were averaged and found to be signi®cantly higher P<
0:001than the average carbon content for horizons 4±
6.
Total carbon data re¯ect a combination of cell bio-mass and exopolymer accumulation associated with the bio®lm. The C-to-N ratio provides a general indi-cation of whether EPS comprises a signi®cant fraction of total carbon, because EPS sugars typically have a higher C-to-N ratio than biomass (Costerton et al., 1978). When the C-to-N ratios from air-dried soil samples from horizons 1±3 of all columns were aver-aged and compared to similar averages for soil from horizons 4±6, the dierences were signi®cant
P<0:001)(Table 1) and they suggest that much of
the total carbon in the upper horizons re¯ects EPS sugar and not biomass.
3.6. Methane oxidation potential
The CH4oxidation potential of soil from the
labora-tory soil columns is presented by horizon in Table 4. Oxidation potential re¯ects the relative size of CH4
-oxidizer populations. The average of CH4 oxidation
potential measures from the top three horizons of all columns signi®cantly exceeded that of the bottom three horizons P<0:001), where O2was more limited
(Table 1).
3.7. Trends among the EPS indicators
In the laboratory columns, horizons with large ac-cumulations of stain were also the horizons with high glucose concentrations and in many cases, the horizons with high resistance to ®ltration. The location of
elev-ated total carbon (Table 1) and CH4oxidation
poten-tial (Table 4), denoting regions of biomass accumulation, also corresponded with regions of high EPS production. Oxygen concentrations in horizons 2 and 5 were 10 and 3%, respectively, suggesting that the lower CH4 oxidation potential and lower EPS
ac-cumulation were related to O2availability.
The measures of glucose, carbon, CH4oxidation
po-tential and ®lterability in soil from the laboratory col-umns were normalized on a 1±10 scale and the relative trends by horizon are shown in Fig. 5. The strength of the relationships between pairs of variables (glucose concentrations, ®lterability, total carbon content and CH4 oxidation potential) was evaluated using
Pear-son's correlation (r). All correlations were statistically signi®cant P<0:05), with correlation coecients of ÿ0.67 for ®lterability and glucose, ÿ0.76 for ®lterabil-ity and carbon, ÿ0.61 for ®lterability and activity, 0.89 for glucose and carbon, 0.94 for glucose and activity and 0.85 for carbon and activity. Glucose concen-trations, total carbon content, and CH4 oxidation
po-tential all had peak values in horizons 1 or 2 and minimum values in horizons 5 or 6 (Tables 1, 3 and 4). Filterability trends were somewhat dierent, with the peak occurring at horizon 3 and high viscosity measures sustained into horizon 4 (Table 2), where values for glucose, total carbon and activity declined.
In the ®eld cores, some of the dierences between the ®lterability of land®ll cores and control cores are likely due to varying soil type. However, the trend toward high glucose concentrations in horizons with low ®lterability suggests that some of the ®lterability dierences are due to EPS. This correspondence is par-ticularly evident in core 3 (Table 2).
Fig. 5. Comparison of trends in ®lterability (ml ®ltrateretained on ®lter after 1 h), glucose concentration (mg kgÿ1 dry soil), total car-bon (%) and relative CH4uptake (ml dÿ1). Measures are the average
values for soil collected from three columns sparged with synthetic land®ll gas for over 2000 h. The values for each parameter were nor-malised so that the minimum value was equal to 0 and the maximum value was equal to 10. The relative measures are plotted by horizon. Actual measurements for total %C, ®lterability, glucose concen-tration and CH4oxidation potential are presented in Tables 1±4,
re-spectively. Table 4
Methane oxidation potential by horizon
Average ml uptake dÿ1a
Horizon after LFG before LFG
0.6920.09
4. Discussion and conclusion
EPS production in LFG-sparged laboratory soil col-umns and in fresh land®ll soil cores was con®rmed. Although EPS is a normal component of bio®lm growth, it was shown here that a substantial quantity of highly viscous polymeric substance was produced in response to CH4exposure.
The deviation of ®lterability pro®les from the other parameters in horizons 3 and 4 (Fig. 5) suggests that soil with the largest EPS accumulation does not necessarily yield the most viscous extract. EPS vis-cosity is related to the amount of cross-linking of the polysaccharide molecules. It can vary as a function of changes in molecular conformations, polymer concen-trations or the in¯uence of polymers from other organ-isms with dierent conformations or branches that enhance cross-links between two disparate polymers (Christensen et al., 1990). Increasing pH has been found to thicken the capsule of one Methylococcus capsulatus strain (Gordienko et al., 1997), suggesting that the pH gradients that formed in the laboratory columns may exert some in¯uence on the polymer vis-cosity in a horizon. Turakhia and Characklis (1988) have proposed that calcium ions in the bio®lm matrix can enhance the cohesiveness of a bio®lm.
Bacteria have also been shown to produce polyhy-droxybutyrate (PHB) granules under high C-to-N nutrient ratios and limited O2(Senior et al., 1972; Lee,
1996), and methanotrophs are well known for their ability to accumulate PHB (Asenjo and Suk, 1986; Nichols and White, 1989). Upon cell death and lysis, PHB, which is also fairly viscous, is released and can accumulate in soil (Dawes et al., 1973). Thus, it is possible that some of the low glucose-high viscosity material detected in the lower horizons may have included polymers other than EPS.
Although the presence of viscous polymer was con-®rmed by a variety of measures, there was no evidence that it caused short-circuiting and reduced CH4
reten-tion times in the columns. Methane residence time was not signi®cantly dierent in soil columns before and after CH4 exposure and bio®lm accumulation. Thus,
the observed reductions in CH4 uptake from peak to
steady state rates could not be attributed to soil pore clogging.
In the adverse conditions of land®ll cover soil, it is plausible that EPS contributes to the sustenance of CH4 oxidizer populations by providing protection
against desiccation or predation or, it may simply be a manifestation of metabolic adaptations to a carbon-rich environment. Whatever the source of its stimu-lation, a consequence of its production may be that it regulates the rate of CH4oxidation by constraining O2
diusion to cells embedded in the bio®lm. A math-ematical model used to test this hypothesis in the
lab-oratory column system described here demonstrated that the observed trends in CH4 oxidation could be
explained by the development of a viscous EPS layer over a base bio®lm layer (Hilger et al., 1999).
Short-term laboratory-scale experiments of land®ll CH4 oxidation may exclude EPS eects if incubations
do not allow for time-dependent bio®lm thickening. EPS accumulation over time may explain why in serum bottle assays, NO3 stimulates initial CH4
oxi-dation but has little eect when added to soil pre-viously exposed to high CH4concentrations for several
months (Hilger et al., 2000). It would be prudent to examine how factors such as soil type, soil compac-tion, climate, nutrient amendments and pH aect the nature and amount of EPS produced. The nature of the samples tested: compacted soil, unconsolidated soil, soil slurry or extracted soil bacteria may also in-¯uence how EPS eects are manifested.
It remains to be shown whether the viscous exopoly-mer accumulation observed in the land®lls sampled for these experiments is a widespread occurrence. If the as-sociation between CH4 emissions and EPS formation
in land®ll covers is common, then simple glucose assays and ®lterability tests may prove practical for detecting, mapping or monitoring CH4leaks.
Acknowledgements
This research was supported by the William States Lee College of Engineering, UNC-Charlotte and a NSF Presidential Faculty Fellowship to MAB. Consul-tations with Richard Veeh and the assistance of Chris-topher Antonucci are gratefully acknowledged.
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