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Methanotrophic Cultures from Landfill Environments: Promise
for Bioremediation of Hazardous Chemicals
The recent explosive population growth in the state of Florida has resulted in a massive increase in the amount
of municipal solid waste (MSW) and associated hazardous chemicals disposed in landfills. As a result of this
rapid loading of landfills, an urgent need exists for controlling landfill environments so that natural
attenuation processes can more efficiently degrade this waste. In a direct response to this need, we are
investigating bioremediation as a possible method for MSW hazardous chemical degradation. We have isolated
and characterized a methanotrophic-heterotrophic mixed culture (GW 60,13') from the Alachua County Landfill
and are comparing these results to a well-defined, previously isolated groundwater mixed culture, MM1. Initial
results from this work show that these Type II methanotrophic cultures exhibit different characteristics but
are capable of degrading representative volatile organic compounds that are prevalent in landfill environments.
In the last decade, the state of Florida has experienced a growth rate of approximately 40%, and projections
of Florida population growth rates into the year 2010 show values that are on average 92% higher than values
for the rest of the country (1). As a result, the municipal solid waste (MSW) and associated hazardous
chemicals generated in Florida each year have increased to over 24 million tons (2). In order to eliminate this
waste, environmental engineers have resorted to landfilling up to 40%, despite efforts to promote recycling
and composting (2). As a result of the rapid loading of landfills, there is an urgent need to control
landfill environments for rapid degradation of this waste before leaching into potential drinking water sources occurs.
The predominant degradation processes in landfill environments are biological and differ depending on the levels
of oxygen present (3). Current landfill design promotes the formation of anaerobic processes (with no
oxygen present), and the primary microorganisms involved in these processes are known as "methanogens."
These microbes are responsible for breaking MSW down to methane, a key component of greenhouse gasses.
The actual amount of methane released into the atmosphere from landfills is approximately 10-70 Tg per year
(4), with a significant portion intercepted by methanotrophs, aerobic microorganisms that reside near
the methanogenic zones and require stable sources of both methane and oxygen (4). Certain
methanotrophs (expressing soluble methane monooxygenase, sMMO) have been suggested to play a role
in transforming a variety of other compounds of environmental interest, including polychlorinated biphenyls
(PCBs) (5,6,7, 8) and trichloroethylene (TCE) (9,10). However, such studies have not been directly targeted to
solving contamination problems specific to landfill environments. Thus, further research is needed to address
our limited understanding of the diversity, distribution, and potential for landfill microorganisms to transform
leachate chemicals into harmless forms in an optimized fashion.
The aims of this study were (i) to isolate a novel methanotrophic-heterotrophic mixed culture from samples
taken from the Alachua County Landfill, (ii) to characterize this mixed culture based on cellular and
colony characteristics and activity, (iii) to compare these characterization results to a well-defined
mixed methanotrophic-heterotrophic culture (MM1) previously isolated from an uncontaminated
groundwater environment. Based on the results reported herein, these two mixed cultures exhibit
divergent characteristics, yet both have been shown to transform representative organic contaminants prevalent
in landfill environments.
MATERIALS AND METHODS
Both solid and liquid media were used for culturing methanotrophic populations expressing sMMO (expressed
under low or no copper concentrations). The liquid media used was based on Whittenbury's nitrate mineral
salts (NMS) media with no copper added (Table 1) (11). Methanotrophic bacteria were isolated on solid plates
using NMS medium and Bactoï¿½ agar (Difco, Detroit, MI), and heterotrophic bacteria were isolated on nutrient
agar (Difco, Detroit, MI).
Chemicals Needed for NMS Preparation Chemical Components in NMS Medium
MgSO4 7H20 (magnesium sulfate) ï¿½ 1.0 g/1.0 Liter
KNO3 (potassium nitrate)ï¿½ 1.0 g/1.0 Liter
CaCI2 (calcium chloride)ï¿½ 0.2 g/1.0 Liter
FeEDTA" 0.1 mL/1.0 Liter
NaMolybdate- 4 H20 0.5 mL/1.0 Liter
Whittenbury Trace Elements (already prepared) 1.0 mL/1.0 Liter
Phosphate Stock Solution (already prepared)* 10.0 mL/1.0 Liter
Vitamin Stock Solution (already prepared)* " 10.0 mL/1.0 Liter
* indicates that these chemicals are added to the media after autoclaving (phase 2)
ï¿½ indicates that the chemical is in solid form
" indicates that the chemical is in liquid form
Isolation and Maintenance of Liquid Landfill Mixed Cultures
To isolate this culture, one gram of a soil sample, taken 13 feet below the landfill surface, was added to 25 mL
of NMS liquid medium in a 250 mL Erlenmeyer flask, equipped with a rubber stopper and a glass wool-packed
filling tube. Headspace was removed from the flasks through the filling tubes using a vacuum pump apparatus,
and an equivalent amount of 99.9% methane (Strate Welding, Jacksonville, FL) was added to achieve a methane:
air ratio of approximately 30:70. The cultures were incubated and shaken at 30 oC and 270 rpm. After
detecting sufficient visible turbidity (~7 days), transfers were prepared using a 10-20% inoculum and repeated
until a stable culture was detected (determined visually on solid medium). Once verified as stable, it was given
the name "GW 60, 13" in reference to the well number and depth of sampling.
Maintenance of Solid cultures
Both MM1 and GW 60,13' cultures were serially diluted and spread plated onto nutrient and NMS agar plates.
NMS plates were incubated under methane and air (30:70) using airtight dessicators (at 300 C), and nutrient
plates were stored in tupperware containers at 300 C, with routine swabbing of the containers with ethanol to
prevent fungal contamination.
Colony and Cellular Morphology Characterization
Characterization of colonies involved visual observations of colony size, overall shape, margin, elevation,
color, transmittance of light and any other noticeable details (i.e., changes in growth over time).
Cellular characterization methods involved Gram and methyl violet staining and scanning electron
microscopy techniques. Samples of both mixed cultures were prepared for scanning electron microscopy by
fixing with Trumps fixative (buffered 1% glutaraldyhyde, 4% formalin), postfixing with 4% oxmium tetroxide,
rinsing with ethanol solutions of increasing concentration, and mounting the dried samples by sputter coating with
a gold/palladium mixture. Prepared samples were then viewed on a Hitachi S-4000 scanning electron microscope.
Measurement of Growth and sMMO Activity
Growth curves, measured using side-armed flasks equipped with the rubber stopper design described
previously, were prepared using a UV/VIS spectrophotometer (Fischer Technical Cpy., Schaumburg, IL) at
a wavelength of 600 nm. sMMO assay involved incubation of suspended cultures with naphthalene
crystals. Suspensions expressing sMMO tested positive by the formation of a purple color upon addition of
Oxygen Uptake Measurements
The oxidation potential of MM1 and GW 60,13' against representative environmental contaminants was
measured using oxygen uptake methods. Rates were measured with an oxygen electrode (YSI Co., Yellow
Springs, OH, USA), mounted onto a 2.0-mL jacketed reaction vessel held at a constant temperature (30C) and
linked to an oxygen analyzer (YSI Co.. Yellow Springs, OH). Data were collected at 10 Hz and converted to
digital input by an A/D converter board (CIA-DAS08-PGL, Computer Boards, Inc, Mansfield, MA, USA), mounted in
an Hewlett Packard 486 computer equipped with Labtech Notebook software (Wilmington, MA, USA). Rates
were normalized to oxygen uptake rates in the presence of methane.
Increasing turbidity in the initial landfill sediment liquid cultures was visible after approximately 7 days of
incubation. Subsequent routine transfers of the stable consortium showed significant growth in approximately 3
days after inoculation. Growth on nutrient agar plates was rapid, with visible colony formation after 1 day.
Isolation of the GW 60,13' methanotroph(s) is still in progress; however, consistent growth appears on NMS plates
3-4 days after streaking.
Table 2 summarizes the characteristics of the GW 60,13' heterotrophic community. Upon streaking onto
nutrient agar, 12 different colonies were initially isolated; however, after repeated transfers onto fresh nutrient
agar plates, only 6 colonies appeared to be distinct from the rest. Whether this was due to the inability to
visually distinguish the colonies together on nutrient plates or to the loss of colonies upon repeated streaking is
not known. Table 3 describes the cellular and colony morphologies and the growth characteristics of these 6
stable colonies. The liquid mixed culture MM1 showed 4 heterotrophs on nutrient agar. Characteristics of
these colonies are summarized in Table 4.
Colony and Cellular Characteristics of GW 60, 13 Heterotrophs
SHAPE COLONY AMOUNT
GRAM CELL COLONY SHAPE COLONY COLONY COLONY L AMOUNT CATALASE
COLONY OF TRANSMITTANCE OF
REACTION SHAPE PIGMENTATION MARGIN ELEVATION CONSISTENCY REACTION
COLONY TO LIGHT GROWTH
1 Cream, Smooth
B White/ cream Circular Convex Mucoid glossy Translucent 1+ +
B and Smooth
Tan - Tan/ dark orange Circular Convex Mucoid glossy Translucent 1+ +
Cream Light orange/ Smooth
+ CB Circular Convex Mucoid glossy Translucent 2+ +
clump cream Entire
White 1 Smooth
B White/ cream Circular Convex Mucoid glossy Translucent 3+ +
White 1 - B Light Yellow Circular? Convex Mucoid glossy Translucent 3+ +
+ B Electric Pink Circular or Umbonate Dry Opaque 3+ +
+ CB Pastel Yellow Circular Irregular Umbonate Dry Opaque 3+ +
C - It. Light orange/ Smooth
B Circular Convex Mucoid glossy Translucent 2+ +
Orange cream Entire
A - beige - B White cream Circular Convex Mucoid glossy Translucent 2+ +
Cloudy - CB White Cream Circular Raised Mucoid glossy Translucent 2+ +
B Tan/ Dark Orange Circular Convex Mucoid glossy Translucent 2+ +
2 - white - B White Cream Circular Convex Mucoid glossy Translucent 2+ +
1+ = growth on 1/3 of plate; 2+ =growth on 2/3 of plate; 3+ = growth on entire plateC= cocci CB= coccobacilli B= bacilli S= spirilla V= vibri
GW 60,13' Heterotrophic Colonies
Number Colony Name Colony Color
1 Yellow-white 1 clump Light yellow
2 Yellow glob Pastel yellow
3 Orange/pink Electric pink
4 Tan/ dark orange
C - It. Orange
5 Light orange/ cream
White 1 clump
1 cream glossy
Colony and Cellular Characteristics of MM1 Heterotrophs
CULTURE GRAM CELL COLONY SHAPE COLONY COLONY COLONY COLONY
NAME REACTION SHAPE PIGMENTATION OL MARGIN ELEVATION CONSISTENCY TOANSMIT
COLONY TO LIGHT
B White cream Circular
B Cream Circular
B Yellow Circular
B Beige Circular
Convex Mucoid glossy Translucent
Mucoid glossy Translucent
Convex Mucoid glossy Translucent
2+= growth on 2/3 of plate; 3+= growth on entire plate
Scanning electron microscopy (SEM) photos (Figures 1A, B, C, and D) reveal the diversity of the mixed GW
60,13' culture as is expected of isolates from environments rich in substrates. The photos show a variety of
cell shapes, sizes, and appendages, including filamentous extensions and flagella. Figures 2A, B, C show
SEM photos of MM1. As is evident, this culture is less diversified than GW 60,13'. Cells in this culture were
typically bacilli and cocci in shape, most with smooth surfaces and convex in nature.
Figure id. SEM Photograph of GW60, 13' (8,000x).
4 - White
3 - Cream
2 - yellow
1 - cream
Figure id. SEM Pnotograpn ot uwoo, 13 (11,uuox).
Figure id. SEM Photograph of GW60, 13' (18,000x).
Figure id. SEM Photograph of GW60, 13' (13,000x).
Figure 2a. SEM Photograph of Strain MM1 (2,000x).
Figure 2b. SEM Photograph of Strain MM1 (5,000x).
Figure 2c. SEM Photograph of Strain MM1 (18,000x).
Figure 3 shows the growth curve obtained for the GW 60, 13' liquid culture grown under a 30:70 methane:air
ratio. The corresponding growth rate for GW 60, 13' under these conditions was calculated to be 0.342 d -1 with
a doubling time of 2 days. Both mixed cultures showed positive expression of the sMMO and were
tentatively characterized as Type II methanotrophs, capable of expression of pMMO or sMMO, depending on
the concentration of copper present in growth medium.
- = [1l/(t2-tl)][I nx2/xl]
~ -342 day
I I I^
0 10 20 30
40 50 60
Figure 3. GW60, 13' Growth Curve.
Oxygen uptake rates were measured at varying concentrations of selected contaminants. Figures 4 and 5
show oxygen uptake curves for MM1 in the presence of biphenyl and GW60, 13' in the presence of
toluene, respectively. As is shown, oxidation activity was shown by each culture with possible inhibition occurring,
as evidenced by the maximum rate followed by a sharp rate decrease.
0 75 150 225 300 375 40 525
Toluene Concentration, PM
Figure 4. Oxygen Uptake Curve for GW60, 13' with Varying Concentrations of Toluene.
u2 * I "----------------------------
Concentrations of Biphenyl.
0 50 100 1S0 2 3IV 3MO
Biphonyl Concentratiou. PM
Figure 5. Oxygen Uptake Plot of Strain MM1 with Varying
DISCUSSION AND CONCLUSIONS
In this paper, we describe a new mixed methanotrophic-heterotrophic culture, tentatively referred to as GW60,
13'. Isolated thirteen feet below the surface of a landfill in the cover soil zone, this culture shows 6
stable heterotrophic populations and one or more methanotrophs. The difficulty in separating the methanotroph
from the heterotrophs is possibly due to the positive interactions that exist between the two types of bacteria
as hypothesized previously by researchers (12). Work is ongoing to elucidate the roles that the
individual methanotrophs play in these mixed cultures.
Upon comparison of the heterotrophic populations isolated from the landfill culture with those isolated from
an uncontaminated groundwater environment, a divergence in the complexity of the mixtures is evident. Whereas
the heterotrophs isolated from GW60, 13' showed a wide variety of cellular and colony characteristics (as shown
in Table 3), those isolated from the groundwater environment were fewer in number (only four) and more similar
in cellular and colony size and shape. This decrease in complexity of populations is expected for cultures derived
from relatively pristine environments in comparison to those derived from environments rich in organic carbon.
Both mixed cultures tested positive for the expression of sMMO, and, therefore, their potential for oxidizing a
broad range of substrates, including aliphatic and aromatic compounds, is high. Initial screening of
the biodegradative activity of these mixed cultures by oxygen uptake experiments demonstrated that the cultures
are capable of transforming aromatic compounds, such as toluene and biphenyl, with possible inhibition
indicating that either the substrate or intermediates formed may be inhibitory to successful bioremediation.
Future work will address more thorough characterization methods for the isolated landfill mixed and pure
cultures, including genetic analysis. Also the optimum conditions (pH, temperature, concentrations of
nutrients, oxygen, and methane) for growth and contaminant degradation will be assessed. Ultimately, we expect
our research to provide a better understanding of the community structure and interactions of
microorganisms present in landfill environments. This knowledge will then be applied to MSW sites as a
possible method for controlling the landfill environment to effect maximum natural bioattenuation of
We thank Scott Whitaker for his time and patience in preparing samples for SEM viewing in the Electron
Microscopy Core Laboratory (UF campus). We also thank Strate Welding for supplying methane and acetylene tanks.
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