Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue4.2000:

Soil Biology & Biochemistry 32 (2000) 457±467
www.elsevier.com/locate/soilbio

Methane oxidation and microbial exopolymer production in
land®ll cover soil
Helene A. Hilger a,*, David F. Cranford a, Morton A. Barlaz b
a

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 di€usion. 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 kgÿ1 dry 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 onethird 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 shortcircuiting due to clogging was not occurring. The data support the hypothesis that EPS impeded oxygen di€usion to an active
bio®lm and limited the extent of methane oxidation. 7 2000 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 CH4 is 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 atmosphere, and the degree to which this occurs and the

* Corresponding author. Fax: +1-704-510-6953.
E-mail address: hhilger@uncc.edu (H.A. Hilger).

conditions that promote it are all under investigation
(Whalen et al., 1990; Jones and Nedwell, 1993; Kjeldsen 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;

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 0 1 - 7

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H.A. Hilger et al. / Soil Biology & Biochemistry 32 (2000) 457±467

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 accumulation of extracellular polymers that either clogs soil
pores and causes short-circuiting or impedes gas di€usion into the cells.
Many bacteria, including CH4 oxidizers, produce
exopolymeric substances (EPS) that can serve as a
source of anchorage. EPS production may also o€er
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 O2 de®ciency (Wrangstadh et al., 1986).
EPS accumulation can alter the metabolism of bacteria embedded in a bio®lm. Composed largely of
polysaccharide (Costerton et al., 1981), a viscous ®lm
can o€er greater resistance to substrate di€using into
the base ®lm (Christensen et al., 1990; Mozes et al.,
1992) and there is evidence that di€usivity 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; Jensen et al., 1991). Chida et al. (1983) described two
polymers produced by a single thermophilic methanotroph. 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 residues. A highly viscous polymer produced by Methylophilus viscogenes is harvested and marketed under the
name of Poly 54 (Leak et al., 1992). It has been

suggested that for methanotrophs in particular, production of a carbon-rich polymer is used as a metabolic
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 production 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 laboratory columns that had been gassed with CH4 for several thousand hours, on fresh soil cores collected from
a land®ll cover, and on soil cores from sites with no
history of CH4 exposure.
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 variety 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 ®lterability 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 CH4 uptake.
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 history of CH4 production. Fresh ®eld core samples were
collected from the Renaissance Park land®ll (Charlotte, NC), which is closed and has been converted to
recreational ®elds. Control cores with exposure to only
atmospheric CH4 (1.7 ml lÿ1) were taken from the University of North Carolina at Charlotte campus.
Fresh cores were collected by driving a 30 cm long
by 5 cm dia pipe into the land®ll cover surface. The
cores were returned to the laboratory and stored at
48C. Cores were mechanically extracted from the tubes
within 24 h of collection, and about 20 cm of soil was
recovered from each core.

H.A. Hilger et al. / Soil Biology & Biochemistry 32 (2000) 457±467

2.3. Soil columns

Four laboratory soil columns were used to measure
CH4 breakthrough 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, overlain 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 fabricated 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 outdoors. After capping, the headspace volume was approximately 5.3 l.

459

hydraulic conductivities (K ). Columns were prepared
with a hydraulic conductivity of 10ÿ5 cm sÿ1.

2.5. Column operation and monitoring
A synthetic land®ll gas (LFG) containing a 50/50
mix of CO2/CH4 was delivered at 3.25  10ÿ7 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 di€used vertically
from the top. Routine monitoring included measurement 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.

2.4. Column ®lling
Soil columns were prepared within 2 d of soil collection. The soil moisture content was adjusted to 15 2
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 preliminary tests to calibrate the compaction to various

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 chromatography (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
cm3 minÿ1. Injector and oven temperatures were maintained 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 concentration measurements.

2.7. Breakthrough curves

Fig. 1. Soil column reactor design.

Initial breakthrough curves were obtained by withdrawing 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 consumption would not confound the results. Hourly
headspace gas monitoring was repeated for 24±30 h,
after which normal air¯ow was re-established.

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H.A. Hilger et al. / Soil Biology & Biochemistry 32 (2000) 457±467

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, ®lterability, 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 bu€er
(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 particles. 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 bu€er. 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 crucible 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

Fig. 2. Methane consumption in four replicate soil columns sparged
with synthetic land®ll gas.

2000). 8-g soil samples adjusted to 1520.5% moisture
content were sealed in 45 cm3 vials. 7 cm3 of headspace 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 throughout, and a distinct pH gradient developed. Fresh soil
pH was 6.3, but soil removed from the top of the reactors 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

A portion of the unused soil slurry prepared for ®lterability 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 Elemental Analyser.
2.13. Methane oxidation potential
The measurement of CH4 oxidation potential has
been described and is summarized here (Hilger et al.,

Fig. 3. Breakthrough curves for methane migration through soil columns initially and after 2400±3300 h of sparging with synthetic land®ll gas. The values shown are the average of measurements from
four replicate columns with standard error bars (in some cases where
errors were small, bars are obscured by data markers). The time represents the elapsed time from initiation of the breakthrough curve
measurement protocol.

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H.A. Hilger et al. / Soil Biology & Biochemistry 32 (2000) 457±467

faster CH4 migration 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 sucient rheological stability
to withstand disturbances imposed during slide preparation.
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 aggregation than upper horizon samples.
Di€erences 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, di€erences 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 trichosporium OB3b, 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 substantiate the potential for a physical distinction
between two forms of polymer in the same methanotroph 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 columns after gassing, soil from columns 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 viscosity and presumably high EPS (Table 2). Soil from
the upper three horizons of columns gassed with CH4
was dicult 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 cm3 hÿ1).
The ®lterability measures of all column or core
horizons within a treatment were averaged for statistical 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 control cores. Land®ll core 3 was much more resistant
…P < 0:001† than the controls but not signi®cantly
di€erent from the land®ll-gassed laboratory column
soil …P ˆ 0:05), con®rming that the polymer accumulation found in the laboratory columns was not an experimental 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
percent (v vÿ1)c

N2

CH4

pHa
mass percent

Total carbonab

Carbon-to-nitrogen ratioa

1
2
3
4
5
6

16.1
22.4
28.2
33.8
38.2
40.5

60.3
53.2
46.2
38.7
32.4
28.3

10.7
14.5
18.3
22.6
26.3
28.9

5.220.08
5.220.03
5.420.04
6.020.01
6.220.05
6.320.05

0.6620.04
0.6520.03
0.6020.02
0.4320.03
0.3420.01
0.3220.03

2220.52
2220.56
2120.27
1420.27
1320.51
1320.82

13.0
9.9
7.2
5.0
3.1
2.3

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.

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H.A. Hilger et al. / Soil Biology & Biochemistry 32 (2000) 457±467

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 column or core horizons within a treatment were averaged, and only horizons from the top 20 cm of the
laboratory columns were considered in comparisons
with ®eld core samples. There were large di€erences 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 laboratory columns gassed with LFG was also signi®cantly
greater …P < 0:001† than the mean glucose concentration in the ®eld core samples from control sites.
There were large di€erences 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 di€erences
may re¯ect relative amounts of CH4 exposure 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 column sparged with air only was not signi®cantly di€erent …P ˆ 0:05† from the glucose content of the control
®eld cores.
The Dubois test is commonly used with glucose colorimetric 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 polymers are present in equal mass, but contain di€erent
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

Fig. 4. Soil sampled from columns sparged with synthetic land®ll gas. The alcian blue dye-stained regions denote the presence of polysaccharide.
(a) Horizon 1 (top): dense polymer completely coats soil and (b) horizon 3: polymer strands separate from soil particles (20 objective).

Table 2
Comparison of ®lterability of soil with or without exposure to land®ll gasa
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

Horizon

ml ®ltrate collected hÿ1 kgÿ1 dry soil

3
7
11
17
22
27

0.3620.07
0.3020.05
0.2220.04
0.3520.04
0.8520.06
1.0420.19
426

1.9220.01
1.6520.00
1.8220.00
1.8820.01
1.7920.06
1.7920.08
3.2

1.08
1.11
1.61
1.71

1.01
0.61
1.18
1.48

0.35
0.20
0.13
0.49

0.90
1.48
1.66
1.77

1.1920.11
1.0120.10
1.1020.08
1.0720.11

38

655

1158

108

38

a

No statistics are shown where measures represent one sample per horizon.
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.
d
Control cores had exposure to atmospheric methane only.
e
See Table 3 for values by horizon.
b

Table 3
Comparison of glucose content in soil with or without exposure to land®ll gasa
Horizon

1
2
3
4
5
6

Depth

Soil column+LFG-1b

(cm)

mg glucose kgÿ1 dry soil

3
7
11
17
22
27

678222
685287
57821.8
30720.0
183240
240225

Soil column+LFG-2b

Soil column+LFG-3b

Land®ll core 1

Land®ll core 2

Land®ll core 3

Land®ll core 4

67520.0
72120.0
588218
21828.7
142216
9621.5

59227.0
623221
37820.2
192211
14428.0
246212

30
73
23
26

648
1123
299
550

746
1608
1176
1101

281
74
58
18

Range of control coresc

H.A. Hilger et al. / Soil Biology & Biochemistry 32 (2000) 457±467

1
2
3
4
5
6
Average glucose all horizons (mg kgÿ1 dry soil)e

(cm)

0±92
12±85
0±106
0±81

a

No statistics are shown where measures represent one sample per horizon.
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.

b

463

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H.A. Hilger et al. / Soil Biology & Biochemistry 32 (2000) 457±467

(after correcting for fresh soil carbon) and the carbonto-nitrogen (C-to-N) ratio in each horizon of soil after
gassing with LFG are presented in Table 1. The carbon 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 <
0:001† than the average carbon content for horizons 4±
6.
Total carbon data re¯ect a combination of cell biomass and exopolymer accumulation associated with
the bio®lm. The C-to-N ratio provides a general indication 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 averaged and compared to similar averages for soil from
horizons 4±6, the di€erences 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 CH4 oxidation potential of soil from the laboratory soil columns is presented by horizon in Table 4.
Oxidation potential re¯ects the relative size of CH4oxidizer 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 O2 was more limited
(Table 1).

ated total carbon (Table 1) and CH4 oxidation potential (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 accumulation were related to O2 availability.
The measures of glucose, carbon, CH4 oxidation potential and ®lterability in soil from the laboratory columns 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 Pearson's correlation (r ). All correlations were statistically
signi®cant …P < 0:05), with correlation coecients of
ÿ0.67 for ®lterability and glucose, ÿ0.76 for ®lterability 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 concentrations, total carbon content, and CH4 oxidation potential 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 di€erent, 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 di€erences 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
di€erences are due to EPS. This correspondence is particularly evident in core 3 (Table 2).

3.7. Trends among the EPS indicators
In the laboratory columns, horizons with large accumulations of stain were also the horizons with high
glucose concentrations and in many cases, the horizons
with high resistance to ®ltration. The location of elevTable 4
Methane oxidation potential by horizon

Horizon

Average ml uptake dÿ1a
after LFG

1
2
3
4
5
6

2.7820.27
2.7320.30
2.7020.10
1.0220.18
0.9720.06
1.3020.02

before LFG
0.6920.09

a

Value is the average of three replicates from column 2 and four
replicates from column 32S.E. Column 2 was sampled after 2800 h
and column 3 after 4100 h.

Fig. 5. Comparison of trends in ®lterability (ml ®ltrate retained on
®lter after 1 h), glucose concentration (mg kgÿ1 dry soil), total carbon (%) and relative CH4 uptake (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 normalised 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 concentration and CH4 oxidation potential are presented in Tables 1±4, respectively.

H.A. Hilger et al. / Soil Biology & Biochemistry 32 (2000) 457±467

4. Discussion and conclusion
EPS production in LFG-sparged laboratory soil columns 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 CH4 exposure.
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 viscosity is related to the amount of cross-linking of the
polysaccharide molecules. It can vary as a function of
changes in molecular conformations, polymer concentrations or the in¯uence of polymers from other organisms with di€erent 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 viscosity 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 polyhydroxybutyrate (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 retention times in the columns. Methane residence time was
not signi®cantly di€erent 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 carbonrich environment. Whatever the source of its stimulation, a consequence of its production may be that it
regulates the rate of CH4 oxidation by constraining O2
di€usion to cells embedded in the bio®lm. A mathematical model used to test this hypothesis in the lab-

465

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 e€ects 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 oxidation but has little e€ect when added to soil previously exposed to high CH4 concentrations for several
months (Hilger et al., 2000). It would be prudent to
examine how factors such as soil type, soil compaction, climate, nutrient amendments and pH a€ect 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 e€ects are manifested.
It remains to be shown whether the viscous exopolymer accumulation observed in the land®lls sampled for
these experiments is a widespread occurrence. If the association 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 CH4 leaks.

Acknowledgements
This research was supported by the William States
Lee College of Engineering, UNC-Charlotte and a
NSF Presidential Faculty Fellowship to MAB. Consultations with Richard Veeh and the assistance of Christopher Antonucci are gratefully acknowledged.

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