Environmental microbial diversity and anthropogenic impact ...
MICROBIAL POPULATION DYNAMICS AND DIVERSITY IN … · 2015-06-11 · MICROBIAL POPULATION DYNAMICS...
Transcript of MICROBIAL POPULATION DYNAMICS AND DIVERSITY IN … · 2015-06-11 · MICROBIAL POPULATION DYNAMICS...
MICROBIAL POPULATION DYNAMICS AND DIVERSITY IN MUNICIPAL SOLID WASTE ANAEROBIC LABORATORY REACTORS
PROJECT REPORT
by
Christopher A. Bareither Dept. of Geological Engineering University of Wisconsin-Madison
Steven J. Fong
Dept. of Bacteriology University of Wisconsin-Madison
Georgia L. Wolfe
Dept. of Bacteriology University of Wisconsin-Madison
Katherine D. McMahon
Depts. of Civil and Environmental Engineering and Bacteriology
Submitted to The University of Wisconsin System
Solid Waste Research Program
August 5, 2009
ii
ABSTRACT This study is directed towards developing relationships between physical and
environmental characteristics of bioreactor landfills, microbial community composition,
and methanogen populations. Anaerobic reactors degrading municipal solid waste are
operated with temperature control and leachate recirculation to optimize biodegradation.
Leachate samples are collected weekly and analyzed for pH, electrical conductivity,
oxidation-reduction potential, and chemical oxygen demand. Biogas produced during
biodegradation is measured volumetrically and composition is assessed for H2, N2, O2,
CO2 and CH4. Microbial community composition is assessed using automated ribosomal
intergenic spacer analysis and methanogen populations are assessed using quantitative
polymerase chain reaction. The reactors have been in operation for approximately 160
d and all exhibit typical leachate chemistry trends of anaerobic degradation. Coupled
with the methane production, the reactors have progressed through the acid phase and
accelerated methane production phase. A DNA extraction methodology was developed
to optimize the concentration of DNA, which involves filtering leachate on a 0.2 µm filter
and extraction with a Mo Bio Powersoil Kit.
iii
TABLE OF CONTENTS Abstract........................................................................................................................... ii
Table of Contents........................................................................................................... iii
List of Figures................................................................................................................. iv
Introduction .....................................................................................................................1
Background.....................................................................................................................2 Municipal Solid Waste Biodegradation .........................................................................2 Environmental Factors Affecting Biodegradation..........................................................3 Microbial Composition and Dynamics in Anaerobic Biodegradation .............................4
Materials and Methods ....................................................................................................6 Municipal Solid Waste..................................................................................................6 Laboratory Anaerobic Reactors....................................................................................7 Leachate Volume and Chemistry .................................................................................8 Gas Production and Chemistry ....................................................................................8
Laboratory Anaerobic Reactor Operation Data................................................................9 Temperature ................................................................................................................9 Leachate Chemistry .....................................................................................................9 Biogas Composition and Methane Production............................................................10
Microbial Method Development .....................................................................................11 DNA Extraction ..........................................................................................................11 Polymerase Chain Reactor Conditions.......................................................................12 Automated Intergenic Spacer Analysis.......................................................................14
Future Work ..................................................................................................................15 Reactor Operation......................................................................................................15 Compression Cells and Scale Comparison ................................................................16 Microbial Analyses .....................................................................................................16
References....................................................................................................................18
iv
LIST OF FIGURES Fig. 1. Gas, leachate, solids, and microbial trends in laboratory-scale
anaerobic reactors (Barlaz et al. 1989). ..........................................................22
Fig. 2. Box-plots of the percent composition of material groups of municipal solid waste samples on a dry mass basis. ......................................................23
Fig. 3. Schematic of the laboratory anaerobic reactors. .............................................24
Fig. 4. Average waste temperature in the laboratory anaerobic reactors. ..................25
Fig. 5. Temporal relationships of leachate chemical parameters in the laboratory anaerobic reactors: (a) pH, (b) oxidation reduction potential, (c) chemical oxygen demand, and (d) electrical conductivity...........................26
Fig. 6. Temporal relationships of methane flow rate and cumulative methane production for (a) Reactor 1, (b) Reactor 2, and (3) Reactor 3. .......................27
Fig. 7. PCR amplification detection of Methanobacteriales and Methanomicrobiales........................................................................................28
Fig. 8. Raw ARISA profiles of the bacterial community in the MSW leachate of Day 75 for (a) Reactor 1, (b) Reactor 2, and (3) Reactor 3. ............................29
1
INTRODUCTION
The landfill industry is currently in transition from the conventional landfill, where
municipal solid waste (MSW) biodegradation is minimized due to limited moisture
addition to the refuse, to the bioreactor landfill, where MSW biodegradation is a primary
objective. Biodegradation is optimized through the increase in moisture content,
increase in temperature, and/or nutrient/microbial seed addition to the refuse (Reinhart
et al. 2002). The most widely used approach is to increase the moisture content through
recirculation of leachate or addition of supplemental liquids (e.g., sewage or industrial
wastewater). Although current bioreactor landfill operation is ad-hoc (Benson et al.
2007), the bioreactor landfill has potential to treat leachate in situ, accelerate waste
stabilization, maximize gas generation, and increase waste settlement (Reinhart et al.
2002; Mehta et al. 2002). In effect, the waste mass serves as an anaerobic treatment
system in which organic carbon in the leachate is converted to landfill gas (Pohland
1975; Reinhart et al. 2002).
In 2006 the United States discarded nearly 169 million tons of MSW to landfills
and incinerators (USEPA 2006). The discarded fraction contained approximately 50%
biodegradable materials (USEPA 2006). Biodegradation these materials is a microbial
mediated process. Organic polymers are broken down through syntrophic relationships
between hydrolytic, fermentative, acetogenic, and methanogenic microorganisms to
ultimately yield carbon dioxide and methane (Barlaz et al. 1989; Levén et al. 2007).
Molecular analyses have identified factors such as age, location, and operational
conditions of landfills, which affect the microbial, and specifically the methanogenic
archaeal, diversity and population (Huang et al. 2002; Huang et al. 2003; Chen et al.
2003a; Laloui-Carpentier et al. 2006). Methanogens are one of the key microbial groups
of interest in landfill research, since they are the primary producers of methane. Limited
studies have assessed the temporal influence on methanogenic diversity and community
2
structure throughout the MSW biodegradation process. Optimization of MSW
biodegradation through the addition of microbial enhanced leachate is a realistic
possibility through state-of-the-art molecular techniques; however, there is a need to first
further our understanding of the microbial dynamics of MSW biodegradation.
This objective of this study is to develop relationships between chemical
characteristics of MSW biodegradation, microbial community composition, and
methanogen populations. Three laboratory anaerobic reactors are operated with
temperature control and leachate recirculation to optimize biodegradation. Leachate
samples are collected weekly for analysis of leachate quality and for DNA extraction.
Biogas production and composition are also monitored. This report summarizes
approximately 160 d of reactor operation, as well as molecular microbial methodologies
developed for DNA extraction, amplification, and analysis.
BACKGROUND
Municipal Solid Waste Biodegradation
Cellulose, hemicellulose, and lignin are the primary organic polymers that
constitute the biodegradable fraction of MSW. Cellulose and hemicellulose constitute
approximately 45-60% of MSW by dry weight, and biodegrade anaerobically to yield
methane and carbon dioxide (Barlaz et al. 1990; Mehta et al. 2002). Lignin, however, is
recalcitrant under anaerobic conditions, and the fraction of MSW dry weight comprised
of lignin (≈ 15% typically) remains stable throughout the life of a landfill (Colberg 1988).
Lignin partially surrounds the cellulose and hemicellulose polymers, reducing complete
biodegradation of cellulose and hemicellulose in MSW to approximately 60% (Barlaz et
al. 1990).
Biodegradation of MSW occurs through syntrophic microbial interactions with
hydrolytic, fermentative, acetogenic, and methanogenic microorganisms (Barlaz et al.
3
1989; Levén et al. 2007), and is typically explained in a series of sequential phases
(Barlaz et al. 1989; Pohland and Kim 1999). Barlaz et al. (1989) used data from nine 2-L
MSW reactors operated at 41 °C with leachate recirculation to generate a four-phase
MSW biodegradation relationship shown in Fig. 1. The aerobic phase is characterized
by depletion of oxygen and transition to anaerobic conditions, whereupon fermentative
bacteria begin hydrolyzing cellulose and hemicellulose to soluble molecules. Hydrolytic
bacteria then convert the soluble molecules to volatile fatty acids (VFAs) (e.g., acetate,
propionate, and butyrate), CO2, and H2 during the anaerobic acid formation phase,
resulting in the accumulation of carboxylic acids. This acid accumulation produces a
decrease in pH and increase in chemical oxygen demand (COD) (Pohland and Kim
1999).
Methanogen population (MPN, Fig. 1) increases, as does the percent
composition of methane, to mark the onset of methanogenesis. During the accelerated
methane production phase, acetogens and methanogens increase the production of
carbon dioxide and methane primarily by utilizing readily available carboxylic acids.
Biodegradation of cellulose and hemicellulose occurs during methane production and
there is an overall increase in pH and decrease in acid concentration and COD (Pohland
and Kim 1999). A peak in the methane production rate marks the transition to the
decelerated methane production phase (Fig. 1), and although gas composition remains
nearly constant, overall gas production decreases. The rate of solids decomposition is
at a maximum during the decelerated methane production phase, and is largely
controlled by the rate of cellulose and hemicellulose hydrolysis.
Environmental Factors Affecting Biodegradation
The variability of biodegradation in full-scale landfills results in a range of
cumulative methane generation from 0.34 to 68 L-CH4/kg-MSW (Barlaz et al. 1990).
4
The primary environmental factors influencing biodegradation include the water content,
moisture movement/recirculation, temperature, and nutrient availability. The water
content of MSW at placement is approximately 20% (wet weight basis), which is below
optimum for anaerobic microorganisms (Themelis and Ulloa 2007). Farquhar and
Rovers (1973) report maximum biogas production (i.e., biodegradation) in MSW reactors
with water contents ranging from 60-80%. Biogas production has also been shown to
increase with leachate recirculation compared to conventional landfilling (Demir et al.
2003). Chugh et al. (1998) operated 200 L reactors at 38 °C with varying recirculation
rate and reported increased daily gas production with more intense recirculation rate.
However, the increase in gas production varied non-linearly with recirculation rate and a
threshold recirculation rate exists that provides a balance of leachate residence time for
microbial population development and leachate flux to remove inhibitory volatile fatty
acids. Farquhar and Rovers (1973) identified an optimum temperature of 37 °C for
biogas production for temperatures ranging from 0-55 °C. Barlaz et al. (1990) reported a
range of optimum temperatures for mesophilic microorganisms to be between 38-42 °C.
Biogas production has also been shown to increase with increasing concentration of
organic solids (Rao and Singh 2004). However, Rao and Singh (2004) identified a
threshold organic solids concentration of approximately 60 g-VS/L, whereupon
biodegradation decreased due to inhibiting effects of increased carboxylic acid
concentrations.
Microbial Composition and Dynamics in Anaerobic Biodegradation
Biodegradation of MSW is a process mediated by a complex community of
microorganisms. For many years, the microbial ecology of MSW biodegradation was a
“black box” that could not be dissected due to methodological limitations. Several more
recent studies have used modern molecular tools to identify factors such as age,
5
location and operational conditions of landfills, which affect the microbial community
structure and diversity.
The most abundant members of the bacterial community (as determined using
molecular techniques) are usually members of the low-GC Gram-positive phylum that
includes Clostridia and Bacillus, and the Bacteriodetes phylum that includes Cytophaga
and Bacteroides (Huang et al. 2003; Huang et al. 2005; Levén et al. 2007). These
organisms likely initiate polymer degradation, which is the initial step in the syntrophic
biodegradation pathway of MSW. In thermophilic systems, members of the
Thermotogae can be present in high numbers (Levén et al. 2007). Methanogenic
Archaea are also critical for biogas production since they catalyze methane formation.
Hydrogenotrophic Methanomicrobiales and acetoclastic Methanosarcinales have been
frequently detected in bioactive landfills (Huang et al. 2003; Laloui-Carpentier et al.
2006).
Methanogens are one of the key microbial groups of interest in bioactive landfill
research, since they are the primary producers of methane. Methanogen population
dynamics has been studied in some detail in other anaerobic systems such as municipal
sewage sludge digesters (McHugh et al. 2003; Conklin et al. 2006), granular sludges
(Collins et al. 2003; Periera et al. 2002), and co-digestion of MSW and sewage (Griffin et
al. 1998; McMahon et al. 2001). The ecology of acetoclastic methanogens (e.g.
Methanosarcina and Methanosaeta) is particularly interesting since these two genera
seem to niche partition between low- and high-acetate concentrations (McMahon et al.
2001; Karakashev et al. 2005; Conklin et al. 2006). Thus, the concentration of acetate in
leachate could have a significant impact on the route of carbon flow through acetate to
CH4 and CO2. These two genera are also known to exhibit different uptake and growth
kinetics (Conklin et al. 2006).
6
A significant limitation in the past investigations of microbial community ecology
is the analysis of a single sample in time. Research has focused on variations of depth in
landfills (Chen et al. 2003a,b), temperature (Levén et al. 2007), degree of waste
stabilization (Calli and Girgin 2005), and leachate recirculation (Huang et al. 2002)
versus conventional landfills (Huang et al. 2003). Although these studies have generated
much new information about the distribution and preferences of anaerobic microbes in
bioactive landfills, much remains to be learned about the temporal variation in
community composition and how this relates to biogas production and waste
stabilization.
MATERIALS AND METHODS
Municipal Solid Waste
Municipal solid waste (MSW) and leachate were collected from Deer Track Park
Landfill, which is a Waste Management site located in Johnson Creek, Wisconsin. The
MSW was approximately 3-4 months old at the time of sampling. A box-plot of the MSW
material composition on a dry mass basis is shown in Fig. 2. The average material
composition reported in Hull et al. (2005) for 1-3 year old MSW in a New Jersey landfill is
also included for comparison. The MSW fine fraction (Fig. 2) is the material passing a
25-mm screen, which contains significant soil and other fine material difficult to visually
identify. With the exception of a smaller fraction of paper/cardboard, likely due to
decomposition, and a larger fraction of miscellaneous, due to increasing difficulty of
visual identification with increasing age of waste, the relative material composition
identified in this study is similar to that in Hull et al. (2005).
The MSW composition of the laboratory anaerobic reactors is composed of the
average of each material group: 17.2% paper/cardboard, 5.3% flexible plastic, 4.8% rigid
plastic, 2.8% textile, 7.3% wood, 7.7% gravel/ceramics/inerts, 0.1% yard waste, 0.2%
7
food waste, 5.1% metal, 0.5% glass, 2.3% miscellaneous, and 46.6% fine fraction. This
composition is the average of a field-scale lysimeter experiment (Breitmeyer et al. 2008),
which will be used to evaluate scale effects on microbial community composition and
population. All MSW, minus the fine fraction, was shredded and passed through a 25-
mm screen prior to placement in the reactors. The composite MSW was blended
thoroughly, hydrated to a water content of approximately 28%, and compacted in five
lifts to an average total unit weight of 7.7 kN/m3.
Laboratory Anaerobic Reactors
A schematic of the laboratory anaerobic reactors is shown in Fig. 2. The reactors
consist of a 0.6-m diameter by 0.9-m tall stainless steel tank. Shredded MSW was
placed in the reactors with a gravel layer at the base for leachate collection and a gravel
layer at the top for leachate distribution. A fine-mesh aluminum screen was placed
between the MSW specimen and drainage gravel to prevent clogging of the effluent port.
Leachate is collected from the effluent port in an intravenous (IV) bag to minimize
exposure to oxygen, and is recirculated via a perforated PVC pipe distribution network
installed in the distribution gravel. Gas produced during MSW biodegradation is
collected in flexfoil SKC gasbags.
The stainless steel tank is heated via two 300-mm x 600-mm flexible silicone
rubber heaters. The heaters are regulated with an on/off relay switch connected to a
Campbell Scientific, Inc. CR23X Micrologger. Omega Engineering, Inc. type-T
thermocouples are used to monitor temperature in the drainage gravel, distribution
gravel, and at four vertically staggered locations in the MSW. Real time temperature
measurements are monitored by the Micrologger via an external thermocouple adjacent
silicone rubber heaters to trigger the on/off relay switch and maintain refuse temperature
near 40 °C for optimal biodegradation.
8
Leachate Volume and Chemistry
Leachate collected from Deer Track Park Landfill is stored in zero head-space
containers at 4 °C to minimize biological activity. Leachate addition to the reactors
began on Day 41 of reactor operation with an initial volume of 10 L. A second 10 L dose
was applied on Day 43. Subsequent leachate addition/recirculation has been executed
in 1 L volumes every 1-2 d. The initial larger dose volumes were applied to seed the
reactors with an active anaerobic microbial culture to initiate biodegradation.
Subsequent smaller volumes were chosen for ease of leachate management. Leachate
collected in the IV bags is consistently recirculated in the reactors, with additional fresh
leachate added if needed to achieve 1 L.
Chemical parameters of the leachate are used to provide a measure of the
biodegradation activity. Effluent samples are collected weekly and analyzed for pH,
electrical conductivity (EC), oxidation-reduction potential (ORP), and chemical oxygen
demand (COD) in accordance with Standard Methods (1999).
Gas Production and Chemistry
Gas produced during biodegradation is measured via water displacement
method in a calibrated carboy submerged in acidified water (pH ~ 3.0). Gas composition
is measured in a Shimadzu Gas Chromatograph (GC-2014) equipped with a flame
ionization and thermal conductivity detector. Relative compositions of hydrogen (H2),
nitrogen (N2), oxygen (O2), carbon dioxide (CO2), and methane (CH4) are calculated with
respect to standard gases (Scott Specialty Gases and Linde Gas Corp).
9
LABORATORY ANAEROBIC REACTOR OPERATION DATA
Temperature
Daily average waste temperatures in the three reactors are shown in Fig. 4.
Daily average waste temperatures were computed as arithmetic means of hourly
measurements recorded at each of the four vertically staggered thermocouples within
the waste specimens. Electrical problems with the relay switch on Reactor 1 caused the
heaters to malfunction and burn out during the first day of operation. Elevated
temperatures during this malfunctioning damaged all thermocouples in Reactor 1 except
the one in the distribution gravel and one within the waste. Subsequent electronic
interference between the damaged thermocouples in Reactor 1 and the datalogger
caused elevated temperatures in reactors 2 and 3 during the first 20 d.
The heaters on Reactor 1 were replaced and electronic interference problems
remedied such that by Day 50 of reactor operation an approximately stable temperature
was achieved in each reactor. The average waste temperature between days 50-150 in
Reactor 1 was 38±2 °C, in Reactor 2 was 34±4 °C, and in Reactor 3 was 34±2 °C.
Leachate Chemistry
Temporal relationships of pH, oxidation reduction potential (ORP), chemical
oxygen demand (COD), and electrical conductivity (EC) for the three reactors are shown
in Fig. 5. Leachate addition to the reactors began on Day 41 of operation, approximately
corresponding to the time of temperature equilibrium. Similar temporal trends for all four
leachate parameters are exhibited in the three reactors. As leachate is added to the
reactors the pH decreases due to accumulation of carboxylic acids. This acid
accumulation is characterized by an increase in COD from 2,400 mg/L in the leachate
inoculum to between 37,000 and 42,000 mg/L by Day 62.
10
The EC relationships show a similar decreasing and subsequent stabilization
trend compared to the COD. A similar phenomenon was reported by Ham and Bookter
(1982), but at lower conductivities ranging from 5 to 20 mS/cm. The ORP is typically
used as an indicator of methanogenesis in MSW research, where a negative ORP,
typically less than 200 mV, is indicative of active methane production (Farquhar and
Rovers 1973). The initial increase in ORP relative to the leachate inoculum (i.e., ORP =
-370 mV) is representative of acid accumulation and inactive methanogenesis. As COD
decreases, pH increases due to removal of the available carboxylic acids, and the ORP
for all three reactors decreases, indicating active methanogenesis.
Biogas Composition and Methane Production
Methane flow rate and cumulative methane production for the three reactors are
shown in Fig. 6. Although the three reactors are operated similarly and exhibit similar
trends in leachate chemistry, methane production is variable. Gas production began
approximately on Day 70 for reactors 1 and 2, whereas gas production began
simultaneously with leachate addition in Reactor 3, approximately Day 41. Gas
composition is similar in all three reactors (data not shown). The percent CH4 increased
during the first 20 d of gas production from approximately 20% to 60%. Between days
80 and 100 the relative CH4 composition stabilized in all three reactors at 61-63%, with
the balance being CO2.
Methane flow rates and cumulative production in Fig. 5 are normalized with
respect to MSW dry mass. The peak CH4 flow rate in Reactor 3 was 0.69 L-CH4/kg-
MSW/d and occurred on Day 86. Peak CH4 flows in reactors 1 and 2 were of similar
magnitude, 0.43 L-CH4/kg-MSW/d in Reactor 1 and 0.53 L-CH4/kg-MSW/d in Reactor 2,
but occurred later in operation due to the lag period of biogas initiation. The lag period
for gas production in Reactor 1 agrees with the leachate chemistry (Fig. 5); however,
11
leachate chemistry in reactors 2 and 3 is very similar and cannot be used to determine
the lag in gas production for Reactor 2.
Cumulative CH4 production has been reported to range from 0.34 to 68 L-
CH4/kg-MSW for full-scale landfills (Barlaz et al. 1990). The broad range of CH4
production is attributed primarily to variability in MSW composition, and secondarily to
variability in environmental conditions. An increase in CH4 production measured in the
laboratory compared to full-scale operations is typical due to increased contact between
microbes, substrates, and necessary growth factors from waste shredding and efficient
leachate recirculation (Barlaz et al. 1990). Biochemical methane potential experiments
are currently in progress to determine the maximum CH4 yield for the MSW used in this
study.
MICROBIAL METHOD DEVELOPMENT
DNA Extraction
Preliminary testing was directed towards developing a methodology to extract
DNA from the leachate fraction of MSW. Techniques to separate the microbial
community from the leachate were centrifugation and vacuum filtration. Centrifugation
involved centrifuging the leachate at 10,000 g for 10 minutes to pelletize the solid matter.
The supernatant was then decanted and the pellet was used for DNA extraction.
Vacuum filtration was conducted on a 0.2 µm filter paper. After filtration the filter paper
cut into 5-mm squares for DNA extraction. Equal 13.25 mL aliquots of fresh leachate
from Deer Track Park landfill were used in this preliminary process.
Extraction of DNA from the centrifuged pellet and the filter paper was completed
by two methods: (1) a traditional phenol-chloroform method with bead-beating and
alcohol precipitation and (2) the Powersoil DNA Kit (MO BIO, Carlsbad, CA). The
Powersoil DNA Kit was used with the manufacturer’s instructions with slight
12
modifications. The bead-beating step described in the instructions was optimized to a
3.5 setting for 3 minutes on a Biospec Bead Beater. Additionally, 2 µL of 50x
ethylenediaminetetraacetic (EDTA) was added to the final DNA solution (100 µL volume)
to prevent DNA degradation prior to storage in a -20 °C freezer.
The total DNA yield and purity was assessed using a Nanodrop spectrometer.
This instrument measures the absorbance of light by a specimen at various
wavelengths. The ratio of absorbance at wavelengths 260 to 280 nm (A260/A280) is used
to assess the purity of the DNA extraction, where A260/A280 = 1.8 indicates pure DNA.
The total DNA yield is reported by the instrument as a concentration (mass of DNA per
volume of solution). A DNA extraction methodology incorporating vacuum filtration on a
0.2-µm filter combined with the Powersoil DNA Kit yielded the highest concentration and
purest DNA sample. This method was used subsequently for all weekly leachate
samples collected from the reactors.
Polymerase Chain Reactor Conditions
Polymerase chain reaction (PCR) is a method to amplify DNA targets of interest
for use in subsequent molecular analyses (e.g., sequencing, ARISA) (Madigan and
Martinko 2006). PCR was performed on individual DNA samples extracted with the
vacuum filtration/Powersoil Kit method described above. PCR was conducted with 24
µL of a master mix solution and 1 µL of sample DNA. The master mix solution contains
10.85 µL H20, 5.0 µL 5x GoTaq Buffer (Promega, Madison, WI), 5.0 µL betaine, 1.5 µL
MgCl2, 0.15 µL GoTaq polymerase (Promega, Madison, WI), 0.5 µL of 4x10 mM dNTP’s,
and 0.5 µL of forward and reverse primers of interest. Thus, final concentrations of the
forward and reverse primers were 0.4 µM in the master mix solution. Concentrations of
MgCl2 and betaine were optimized using concentration gradients run through the PCR
amplification process. PCR products were then analyzed with gel electrophoresis on a
13
1% agarose gel and viewed with a FOTODYNE System UV Transilluminator (Harland,
WI). For example, betaine was optimized by running a concentration gradient between
0.5 M and 2.0 M with 0.5 M increments. Betaine is used to prevent primer dimerization,
which leads to non-specific amplification.
PCR was performed in an Eppendorf Mastercycler (Eppendorf AG, Hamburg,
Germany) with the following steps: initial denaturing at 95°C for 5 minutes, 35 cycles of
denaturation at 95°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for
1.5 minutes, and final extension at 72°C for 10 minutes. Thermocycler settings were
adapted from Staley (2009), but optimized and revised due to differing thermocyclers.
Primers targeting four methanogenic orders (Methanococcales,
Methanobacteriales, Methanomicrobiales, and Methanosarcinales) and two families
(Methanosarcinaceae and Methanosaetaceae) were selected from Yu et al. (2005) to
quantify the methanogenic community in MSW leachate. These primers will be used in
a quantitative PCR (qPCR) procedure currently being developed; however, preliminary
qualitative screening of the primers was conducted to optimize PCR thermocycler
settings.
An image of the gel electrophoresis for the Methanobacteriales and
Methanomicrobiales primer sets is shown in Fig. 7. Bands in rows 1 and 10 correspond
to a ladder, which is a mixture of fragments of known basepairs to compare with PCR
products. Methanobacteriales primer sets were used to screen three leachate samples
and a positive detection is indicated by the single bands shown in rows 3, 4, and 5.
Methanomicrobiales primer sets were also used to screen three leachate samples and
showed positive detection in all three (rows 7-9 in Fig. 7). Methanobacteriales was
detected with primers MBT857f (5’–CGWAGGGAAGCTGTTAAGT) and MBT1196R (5’-
TACCGTCGTCCACTCCTT) and Methanomicrobiales was detected with primers
MMB282F (5’–ATCGRTACGGGTTGTGGG) and MMB832R (5’–
14
CACCTAACGCRCATHGTTTAC). Positive detection of Methanosarcinaceae was also
achieved with primers Msc380F (5’ – GAACCGYGATAAGGGGA) and Msc828R (5’ –
TAGCGARCATCGTTTACG) (Yu et al. 2005).
Automated Intergenic Spacer Analysis
Automated ribosomal intergenic spacer analysis (ARISA) fingerprints provide a
unique snapshot of a bacterial community, with taxa inferred from the base pair length of
their variable 16S-ITS-23S region (Fisher and Triplett 1999). The ITS region is amplified
using a fluorescently tagged forward primer, specific to either bacteria or archaea, and
capillary electrophoresis is used to separate fragments by length while recording the
fluorescence intensity of each fragment. ARISA profiles show a range of peaks, of
increasing length and varying height. The height of ARISA fluorescence peaks at a
given base pair length is a proxy for the relative abundance of that taxon, or “Operational
Taxonomic Unit” (OTU). ARISA profiles from bacterial or archaeal DNA can be
compared across samples to explore changes in presence and abundance of particular
OTUs. This process allows observations to be made regarding the dynamics of the
bacterial or archaeal community. Changes in community profiles can be correlated to
environmental parameters using multivariate statistics.
The intergenic spacer of the 16S-ITS-23S rRNA operon was PCR amplified and
analyzed using ARISA essentially as described in Fisher and Triplett (1999), with minor
modifications as described elsewhere (Shade et al 2007). 1 µl of extracted leachate
DNA was used as a template for 30 cycles of PCR with GoTaq Flexi DNA polymerase
(Promega Corporation, Madison, WI, USA), performed on an Eppendorf Mastercycler
(Eppendorf AG, Hamburg, Germany). Bacterial-specific 1406 forward and universal 23S
reverse primers were used. The 1406F primer was tagged with a fluorescent dye, 6-
15
FAM, on the 5’ end, enabling detection of the amplified product with capillary
electrophoresis on an ABI PRISM 3730 DNA analyzer (Applied Biosystems, Foster City,
CA, USA).
Raw ARISA profiles of the leachate bacterial community collected from each of
the three reactors on Day 75 are shown in Fig. 8. Raw community profiles were size-
calibrated with an internal standard and examined for quality control using GeneMarker
(SoftGenetics, PA, USA). Fragment length increases positively along the x-axis, and
fluorescence intensity increases positively along y-axis. The height of the fluorescence
peak is an indirect measure of abundance, where abundance increases with peak
height. Many OTUs are present in high relative abundance, as expected based on
previous studies showing diverse bacterial communities in bioreactor leachate (Huang et
al. 2005; Levén et al. 2007).
FUTURE WORK
Reactor Operation
The three laboratory anaerobic reactors will be disassembled intermittently
during the next 6 months of operation. Reactor 2 will be disassembled at the end of
June 2009, Reactor 1 will be disassembled in September 2009, and Reactor 3 will be
disassembled in December 2009. Solid samples of the degraded refuse will be
extracted from the reactors and processed for microbial analysis following a phosphate
buffer protocol described in Staley (2009). An assessment of the microbial community
based solely on the leachate fraction can be skewed towards planktonic members more
adapted to a liquid environment, and a more complete characterization of the microbial
community of MSW should account for both the leachate and solids fractions (Staley
2009).
16
Compression Cells and Scale Comparison
Municipal solid waste with the same composition, but varying particle size, as the
laboratory anaerobic reactors is being tested in three varying sized compression cells as
part of a larger project. The compression cells have diameters of 64, 100, and 300-mm
and are all equipped with similar temperature control and gas and liquid management
capabilities as the reactors. Additionally, the compression cells have stress control and
are capable of generating stresses in the range of full-scale landfills (e.g., up to 400
kPa). Leachate will be recirculated in these compression cells are liquid addition rates
typical of full-scale bioreactor landfills (Bareither et al. 2008). Leachate samples will be
collected from these compression cells on a weekly basis for leachate chemistry and
microbial analysis.
In accompaniment to the laboratory experiments, a field-scale lysimeter
experiment is being conducted to assess hydraulic and mechanical at near full-scale
bioreactor conditions (Breitmeyer et al. 2008). Leachate samples are being collected
and processed for leachate chemistry and microbial community composition and
methanogen population. Data from this field-scale project will provide an invaluable
comparison to the applicability of simulating bioreactor operations at laboratory-scale.
Microbial Analyses
ARISA will also be performed on all samples using archaeal-specific primers and
the same methods of analysis used on the bacterial ARISA to generate archaeal
community data. Assignment of peaks in community profiles and standardization
between runs will be performed using R v2.7 statistical software (http://cran.r-
project.org/) and a script used in Kara and Shade (2009). Briefly, the
algorithm calibrates profile data to an internal size standard, and then assigns peaks to
OTU bins (window of base pair size in which a given ITS fragment may occur). These
17
bins will be manually determined in Genemarker, based on an overlay of all
community profiles. Fluorescence will be used as a proxy for the relative abundance of
an OTU in the community profile. Bray-Curtis similarity indices will be used to observe
patterns through time in reactor bacterial and archaeal communities, and analysis of
similarity (ANOSIM) will be used to rigorously test for differences between reactors and
between reactor communities at various stages of the decomposition process.
Correspondence Analysis (CA) will be used to search for patterns in multi-dimensional
ordinations of the data and to link environmental variables, such as pH or CH4 flow rate,
to the observed variation in bacterial and archaeal communities. Clone libraries will be
constructed to link ARISA OTUs to known taxa by comparing sequences to public
databases (e.g. NCBI Genbank and the Ribosomal RNA Database Project). This will
allow exploration of variations in functional microbial communities through time and in
response to changing environmental parameters.
18
REFERENCES
Bareither, C. A., Benson, C. H., Barlaz, M. A., and Morris, J. W. F. (2008) Performance of North American bioreactor landfills, Office of Research and Development, US Environmental Protection Agency, Cincinnati, Ohio. IN REVIEW.
Barlaz, M. A., Schaefer, D. M., and Ham, R. K. (1989). Bacterial population development
and chemical characteristics of refuse decomposition in a simulated sanitary landfill, Applied and Environmental Microbiology, 55(1), 55-65.
Barlaz, M. A., Ham, R. K., and Schaefer, D. M. (1990). Methane production from
municipal refuse: a review of enhancement techniques and microbial dynamics, Manual for Environmental Microbiology, American Society of Microbiology, Washington, D.C., 541-557.
Benson, C. H., Barlaz, M. A., Lane, D. T., and Rawe, J. M. (2007), Practice review of five
bioreactor/recirculation landfills, Waste Management, 27, 13-29. Breitmeyer, R. J., Bareither, C. A., Benson, C. H., Edil, T. B., and Barlaz, M. A. (2008).
Field-scale lysimeter experiment to study hydrologic and mechanical properties of municipal solid waste, Proceedings, Global Waste Management Symposium, Penton Media, Orlando, 1-11.
Calli, B. and Cirgin, E. (2005). Microbial analysis of leachate using fluorescent in situ
hybridization (FISH) technique to evaluate the landfill stability, Fresenius Environmental Bulletin, 14(8), 737-745.
Chen, A., Imachi, H., Sekiguchi, Y., Ohashi, A., and Harada, H. (2003a), Archaeal
community compositions at different depths (up to 30 m) of a municipal solid waste landfill in Taiwan as revealed by 16S rDNA cloning analyses, Biotechnology Letters, 25, 719-724.
Chen, A., Udea, K., Sekiguchi, Y., Ohashi, A., and Harada, H. (2003b), Molecular
detection and direct enumeration of methanogenic Archaea and methanotrophic Bacteria in domestic solid waste landfill soils, Biotechnology Letters, 25, 1563-1569.
Chugh, S., Clarke, W., Pullammanappallil, P., and Rudolph, V. (1998). Effect of
recirculated leachate volume on MSW degradation, Waste Management & Research, 16(6), 564-576.
Colberg, P.J. (1988). Anaerobic microbial degradation of cellulose, lignin, oligolignols,
and monoaromatic lignin derivatives, In Biology of Anaerobic Microorganisms, A. J. B. Zehnder (ed), Wiley-Liss, New York, 333-372.
Conklin, A., Stensel, H.D., and Ferguson, J. (2006), Growth kinetics and competition
between Methanosarcina and Methanosaeta in mesophilic anaerobic digestion. Water Environment Research, 78, 486-496.
Collins, G., Woods, A., McHugh, S., Carton, M. W., and O’Flaherty, V. (2003). Microbial
community structure and methanogenic activity during start-up of psychrophilic
19
anaerobic digesters treating synthetic industrial wastewaters, FEMS Microbiology Ecology, 46(2), 159-170.
Demir, A., Ozkaya, B., and Bilgili, M. S. (2003). Effect of leachate recirculation on
methane production and storage capacity in landfill, Fresenius Environmental Bulletin, 12(1), 29-38.
Farquhar, G. J. and Rovers, F. A. (1973). Gas production during refuse decomposition,
Water, Air, and Soil Pollution, 2(4), 483-495. Griffin, M. E., McMahon, K. D., Mackie, R. I., and Raskin, L. (1998). Methanogenic
population dynamics during start-up of anaerobic digesters treating municipal solid waste and biosolids, Biotechnology and Bioengineering, 57(3), 342-355.
Ham, R. K. and Bookter, T. J. (1982). Decomposition of solid waste in test lysimeters,
Journal of the Environmental Engineering Division, ASCE, 108(EE6), 1147-1170. Huang, L., Zhou, H., Chen, Y., Lou, S., Lan, C., and Qu, L. (2002). Diversity and
structure of the archaeal community in the leachate of a full-scale recirculating landfill as examined by direct 16S rRNA gene sequence retrieval, FEMS Microbiology Letters, 214, 235-240.
Huang, L., Chen, Y., Zhou, H., Lou, S., Lan, C., and Qu, L. (2003). Characterization of
methanogenic Archaea in the leachate of a closed municipal solid waste landfill, FEMS Microbiology Ecology, 46(2), 171-177.
Huang, L. N., Zhu, S., Zhou, H., and Qu, L. H. (2005). Molecular phylogenetic diversity
of bacteria associated with the leachate of a closed municipal solid waste landfill. FEMS Microbiology Letters, 242(2), 297-303.
Hull, R. M., Krogmann, U., and Strom, P. F. (2005). Composition and characteristics of
excavation materials from a New Jersey landfill, Journal of Environmental Engineering, 131(3), 478-490.
Kara, E. and Shade, A. (2009). Temporal dynamics of South End tidal creek (Sapelo
Island, Georgia) bacterial communities, Applied and Environmental Microbiology, 75(4), 1058-1064.
Karakashev, D., Batstone, D. J., and Angelidaki, I. (2005). Influence of environmental
conditions on methanogenic compositions in anaerobic biogas reactors, Applied and Environmental Microbiology, 71(1), 331-338.
Laloui-Carpentier, W., Li, T., Vigneron, V., Mazéas, L., and Bouchez, T. (2006)
Methanogenic diversity and activity in municipal solid waste landfill leachates, Antonie van Leeuwenhoek, 89, 423-434.
Levén, L., Eriksson, A. R. B., Schnürer, A. (2007). Effect of process temperature on
bacterial and archaeal communities in two methanogenic bioreactors treating organic household waste, FEMS Microbial Ecology, 59, 683-693.
20
Madigan, M. T. and Martinko, J. M. (2006). Brock Biology of Microorganisms, 11th Ed., Pearson Prentice Hall, Upper Saddle River, New Jersey.
McHugh, S., Carton, M., Mahony, T., and O'flaherty, V. (2003). Methanogenic population
structure in a variety of anaerobic bioreactors. FEMS Microbiology Letters, 219, 297-304.
McMahon, K. D., Stroot, P. G., Machie, R. I., and Raskin, L. (2001). Anaerobic
codigestion of municipal solid waste and biosolids under various mixing conditions-II: microbial population dynamics, Water Research, 35(7), 1817-1827.
Mehta, R., Barlaz, M. A., Yazdani, R., Augenstein, D., Bryars, M., and Sinderson, L.
(2002), Refuse decomposition in the presence and absence of leachate recirculation, Journal of Environmental Engineering, 128(3), 228-236.
Pereira, M.A., Roest, K., Stams, A.J.M., Mota, M., Alves, M., and Akkermans, A.D.L.
(2002), Molecular monitoring of microbial diversity in expanded granular sludge bed (EGSB) reactors treating oleic acid. FEMS Microbiology Ecology, 41, 95-103.
Pohland, F. G. (1975) Sanitary landfill stabilization with leachate recycle and residual
treatment, EPA Grant No. R-801397, U.S. EPA, National Environmental Research Center, Cincinnati.
Pohland, F. G. and Kim, J. C. (1999). In situ anaerobic treatment of leachate in landfill
bioreactors, Water Science and Technology, 40(8), 203-210. Rao, M. S. and Singh, S. P. (2004). Bioenergy conversion studies of organic fraction of
MSW: kinetic studies and gas yield-organic loading relationships for process optimization, Biosource Technology, 95(2), 173-185.
Reinhart, D. R., McCreanor, P. T., and Townsend, T. (2002), The bioreactor landfill: Its
status and future, Waste Management and Research, 20(2), 162-171. Shade, A. L., Kent, A. D., Jones, S. E., Newton, R. J., Triplett, E. W., and McMahon, K.
D. (2007). Inter-annual dynamics and phenology of bacterial communities in a eutrophic lake, Limnology and Oceanography, 52(2), 487-494.
Staley, B. (2009). Environmental and spatial factors affecting microbial ecology and
metabolic activity during the initiation of methanogenesis in solid waste, PhD Thesis, North Carolina State University, Raleigh, North Carolina.
Standard Methods (1999). Standard methods for the examination of water and
wastewater, Clescerl, L. S., Greenberg, A. E., and Eaton, A. D. eds., 20th Ed., American Public Health Association, Washington, D.C.
Themelis, N. J. and Ulloa, P. A. (2007). Methane generation in landfills, Renewable
Energy, 32(7), 1243-1257. USEPA (2006). Municipal solid waste generation, recycling, and disposal in the United
States: facts and figures for 2006, United States Environmental Protection Agency, Washington, D.C.
21
Yu, Y., Lee, C., Kim, J., and Hwang, S. (2005). Group-specific primer and probe sets to
detect methanogenic communities using quantitative real-time polymerase chain reaction, Biotechnology and Bioengineering, 89(6), 670-679.
22
Fig. 1. Gas, leachate, solids, and microbial trends in laboratory-scale anaerobic reactors (Barlaz et al. 1989).
23
0
10
20
30
40
50
60
Paper/Cardboard
FlexiblePlastic
RigidPlastic
Textile Wood Gravel/Ceramics/
Inerts
YardWaste
FoodWaste
Metal Glass Misc. FineFraction
Pe
rcen
t C
om
positio
n (
%)
- D
ry M
ass B
asis
Average in Hull et al. (2005)for 1-3 yr old MSW from NJ
Fig. 2. Box-plots of the percent composition of material groups of municipal solid waste samples on a dry mass basis.
24
0.6 m
0.9 m
Gravel
drainage
Screen
Shredded
MSW
Perforated
stainless steel
plate
Liquid
distribution
system
Influent tube
Effluent port
Gas outlet
Stainless steel
tank
Gravel
Thermocouple
temperature probes
Silicone
rubber
heater
Position transducer
Fig. 3. Schematic of the laboratory anaerobic reactors.
25
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120 140 160
Reactor 1Reactor 2Reactor 3
Ave
rag
e T
em
pe
ratu
re (
oC
)
Elapsed Time (d)
Reactor 1 heatersburned out
Heaters off
Heaters on
Fig. 4. Average waste temperature in the laboratory anaerobic reactors.
26
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
40 60 80 100 120 140 160
Reactor 1Reactor 2Reactor 3
pH
Leachate InoculumpH = 8.0 (a)
-350
-300
-250
-200
-150
-100
-50
0
50
40 60 80 100 120 140 160
Reactor 1Reactor 2Reactor 3O
xid
atio
n R
eduction P
ote
ntial (m
V)
Leachate InoculumORP = -370 mV (b)
0
10
20
30
40
50
40 60 80 100 120 140 160
Reactor 1Reactor 2Reactor 3
Ch
em
ica
l O
xyg
en
De
ma
nd
x 1
00
0 (
mg
/L)
Elapsed Time (d)
Leachate InoculumCOD = 2400 mg/L
(c)
20
25
30
35
40
40 60 80 100 120 140 160
Reactor 1Reactor 2Reactor 3
Ele
ctr
ica
l C
ondu
ctivity (
mS
/cm
)
Elapsed Time (d)
Leachate InoculumEC = 25.7 mS/cm
(d)
Fig. 5. Temporal relationships of leachate chemical parameters in the laboratory anaerobic reactors: (a) pH, (b) oxidation reduction potential, (c) chemical oxygen demand, and (d) electrical conductivity.
27
0.0
0.1
0.2
0.3
0.4
0.5
0
2
4
6
8
10
12
14
40 60 80 100 120 140 160 180
CH4 Flow Rate Cummulative CH
4
CH
4 F
low
Rate
(L
-CH
4/k
g-M
SW
/d)
Cu
mm
ula
tive
CH
4 (L-C
H4 /k
g-M
SW
)(a)
Reactor 1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0
1
2
3
4
5
40 60 80 100 120 140 160 180
CH
4 F
low
Ra
te (
L-C
H4/k
g-M
SW
/d)
Cu
mm
ula
tive
CH
4 (L-C
H4 /k
g-M
SW
)
(b)
Reactor 2
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
5
10
15
20
40 60 80 100 120 140 160 180
CH
4 F
low
Rate
(L
-CH
4/k
g-M
SW
/d)
Cu
mm
ula
tive
CH
4 (L-C
H4 /k
g-M
SW
)
Elapsed Time (d)
(c)
Reactor 3
Fig. 6. Temporal relationships of methane flow rate and cumulative methane production
for (a) Reactor 1, (b) Reactor 2, and (3) Reactor 3.
28
Fig. 7. PCR amplification detection of Methanobacteriales and Methanomicrobiales.
Methanobacteriales Methanomicrobiales
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
29
Fig. 8. Raw ARISA profiles of the bacterial community in the MSW leachate of Day 75 for (a) Reactor 1, (b) Reactor 2, and (3) Reactor 3.
(a)
(b)
(c)