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The Pennsylvania State University
The Graduate School
School of Science, Engineering, and Technology
IMPACT OF TRICLOSAN ON GENETIC DIVERSITY IN ARTIFICIAL GREEN ROOF
MICROBIAL COMMUNITIES
A Thesis in
Environmental Pollution Control
by
Lauren K. Mehalik
© 2015 Lauren K. Mehalik
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Master of Science
May 2015
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The thesis of Lauren K. Mehalik was reviewed and approved* by the following:
Katherine H. Baker
Associate Professor of Environmental Microbiology
Thesis Adviser
Shirley E. Clark
Associate Professor of Environmental Engineering
Head of the Department of Environmental Pollution Control
Daniel M. Borsch
Lecturer of Biology
*Signatures are on file in the Graduate School
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Abstract
Low-impact development (LID) is a cost effective engineering strategy using natural
features to manage stormwater runoff. Green roofs, vegetated rooftops, are an emerging LID
strategy that not only manage stormwater runoff, but also minimize energy costs, improve
aesthetics, provide wildlife habitats, and reduce the urban heat index. Recycled and reused
materials offer additional improvements to LID techniques by repurposing waste diverted from
landfills. Waste tires can be shredded to provide an artificial growth medium on green roofs. LID
strategies can be further enhanced by reusing greywater (GW) to irrigate an artificial growth
medium of recycled rubber supplemented with compost (RM). Artificial soils are less expensive
and more lightweight than commercially available soils, better accommodating the
weight-bearing limit of buildings. Additionally, GW reuse helps to conserve potable water,
reducing water scarcity. GW often contains triclosan, an antimicrobial found in personal care
products that can select for resistant organisms. Previous studies support the use of RM, but the
impact of greywater and triclosan on microbial communities in RM has yet to be investigated. In
this study, the effects of greywater irrigation on microbial community genomic and phenotypic
diversity were compared in RM and commercially available green roof media (CM). A mixture
of ryegrass and Kentucky bluegrass were grown in each medium and watered with either GW or
greywater with triclosan (GWT). Biolog EcoPlates™ and soil microbial DNA, characterized
through pyrosequencing, were used to analyze microbial diversity. The Biolog EcoPlates™
suggest that the medium, independent of triclosan, has some effect on microbial physiological
diversity. Pyrosequencing results agree with the Biolog plates, showing little change in genomic
diversity after GWT irrigation.
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TABLE OF CONTENTS
List of Figures ................................................................................................................................. v
List of Tables ................................................................................................................................. vi
Acknowledgements ....................................................................................................................... vii
Chapter 1: Introduction .................................................................................................................. 1
1.1. Green Roofs ......................................................................................................................1
1.2. Waste Tires .......................................................................................................................2
1.3. Greywater ..........................................................................................................................2
1.4. Triclosan ...........................................................................................................................3
1.5. Microorganisms ................................................................................................................4
1.6. Biolog EcoPlates™ ...........................................................................................................5
1.7. Soil DNA Extraction .........................................................................................................5
Chapter 2: Materials and Methods .................................................................................................. 7
2.1 Pots, Media, and Planting .................................................................................................7
2.2 Watering ............................................................................................................................7
2.3 Biolog EcoPlate™ Analysis .............................................................................................8
2.4 DNA Extraction ................................................................................................................8
2.5 DNA Quantification ..........................................................................................................9
2.6 Pyrosequencing .................................................................................................................9
Chapter 3: Results and Discussion ................................................................................................ 10
Chapter 4: Conclusion................................................................................................................... 22
References ..................................................................................................................................... 23
v
List of Figures
Figure 1: Triclosan .......................................................................................................................... 3
Figure 2: Growth Trays ................................................................................................................. 10
Figure 3: Shannon Diversity Indices of Biolog EcoPlates™ ........................................................ 11
Figure 4: Early Recycled GW ....................................................................................................... 13
Figure 5: Late Recycled GW ........................................................................................................ 14
Figure 6: Early Recycled GWT .................................................................................................... 15
Figure 7: Late Recycled GWT ...................................................................................................... 16
Figure 8: Early Commercial GW .................................................................................................. 17
Figure 9: Late Commercial GW ................................................................................................... 18
Figure 10: Early Commercial GWT ............................................................................................. 19
Figure 11: Late Commercial GWT ............................................................................................... 20
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List of Tables
Table 1: Composition of Synthetic Greywater ............................................................................... 8
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Acknowledgements
This research would not have been possible without the guidance of Dr. Baker, the use of
her lab, and the IV drip of coffee. Thank you for always being there. Dr. Clark, thanks for always
having time for my last minute questions. Dr. Borsch, thank you for your guidance. Jill Felker,
thanks for coming to the rescue in my moments of grasshopper need. To my parents, Cathy and
George, I would like to extend thanks for all of the support and encouragement along the way.
Mom, this one’s for you.
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Chapter 1: Introduction
1.1. Green Roofs
Vegetated rooftops are gaining worldwide popularity. A low-impact development (LID)
strategy, green roofs provide green spaces in urban areas. Aside from their aesthetic values,
green roofs also reduce heating and cooling costs while benefiting the environment. Green roof
installations transform impervious landscapes into living systems that filter air and purify water
while providing a habitat for insects and wildlife. Concrete buildings and roadways prevent
rainwater from soaking into the ground. Instead, rainwater flows into storm drains and sewer
systems, flushing bacteria and pollutants into streams. Green roof vegetation absorbs rainwater
and lessens the amount of polluted water in aquatic habitats (Happe 2005, Oberndorfer et al.
2007, USEPA 2003).
To prevent roof collapse, lightweight media is used on green roofs instead of the soil
typically used in ground-level gardens. The lightweight media consists of inorganic (sand, brick,
expanded shale, or clay) and organic (peat, compost, or biosolids) mixtures (Oberndorfer et al.
2007, Simmons et al. 2008, Gregoire and Clausen 2011, Panayiotis 2003). Inorganic matter
provides support for plant roots while the organic matter helps to provide nutrients necessary for
plant growth and survival.
Green roofs can be either intensive or extensive, depending on the depth of growth
medium and type of vegetation that can be sustained. Intensive green roofs contain substrate
layers greater than 20 cm deep, supporting vegetation with deeper root systems. Intensive roofs
mimic typical ground-level gardens, supporting a wide range of vegetation from grasses to trees.
In urban areas where parks or gardens are scarce, intensive green roofs provide space for both
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recreation and food-growing needs. Conversely, extensive green roofs contain a shallower
growth medium (< 20 cm) and are not used for public recreation. Shallow medium depths
support low-growing vegetation, including mosses, grasses, or smaller, stress-tolerant plants,
such as sedums. Extensive green roofs are lightweight and require little maintenance
(Oberndorfer et al. 2007).
1.2. Waste Tires
Tires are non-degradable and can serve as mosquito breeding grounds if not disposed of
properly. By weight, rubber makes up about 40% of a tire. Other components include steel and
fluff that provide structure and support to the tire. The rubber is a blend of natural and synthetic
rubbers (Bridgestone 2007). Instead of contributing to landfills, waste tires can be recycled and
used for other purposes, such as fuel, construction materials, or artificial reefs (Lin et al. 2008,
USEPA 2013).
Waste tire size reduction is achieved by shredding the whole tire into smaller pieces.
During the liberation process, steel and fibers are removed from the shredded rubber. The final
step in processing scrap tires is classification, where the size of rubber aggregates produced
through the shredding and liberating steps are further reduced. Larger pieces of rubber return to a
shredder to be reduced to smaller pieces. Screens and mesh near the end of the process separate
the end products by size. Tires can be reduced to small 1- or 2-inch chips, or they can be ground
into smaller crumbs or a fine powder (USEPA 2010). Lightweight and less dense than traditional
soil, crumb rubber can be used as a green roof medium (Solano et al. 2012, Mehalik 2013).
1.3. Greywater
Approximately 50-80% of used residential wastewater is categorized as greywater.
Greywater includes all used water in the home, excluding toilet water, and is generated from
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dishwashing, clothes washing, and bathing. An alternative to sending greywater to wastewater
treatment plants is to reuse the water. Greywater can be recycled for lawn irrigation, conserving
clean drinking water in areas where water is scarce (Salsedo 2011).
1.4. Triclosan
Figure 1: Triclosan (obtained from http://www.chemspider.com/Chemical-Structure.5363.html)
Chemical and physical properties (NCBI 2014)
IUPAC Name 5-chloro-2-(2,4-dichlorophenoxy)phenol
CAS # 3380-34-5
Molecular Weight 289.54178 g/mol
Molecular Formula C12H7Cl3O2
State at Room Temperature White to off-white crystalline powder
Solubility In water, 10 mg/L at 20°C
Melting Point 55-57°C
Boiling Point 120°C
Vapor Pressure 4.6 x 10-6
mm Hg at 20°C
Log KOW 4.76
pKa 7.9
Henry’s Law Constant 2.1 x 10-8
atm-cm m/mole at 25°C
Triclosan (TCS) is an antibacterial agent found in soaps, detergents, plastics, personal
care products (PCP), fabrics, medical devices, and pesticides (APUA 2011). In Negahban-Azar’s
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(et al. 2012) study on the fate of greywater constituents, triclosan was among the antimicrobials
found within the top 30 cm of soil. An octanol-water partitioning coefficient of 4.76 suggests
that triclosan is hydrophobic and less likely to leech into groundwater; it will sorb to organic
matter in the soil.
At lower concentrations, triclosan is bacteriostatic, but at higher concentrations it can
disrupt cell walls and serve as a bactericide. Triclosan can select for resistant organisms through
several mechanisms, including efflux pumps and various enzymes. Some microorganisms utilize
efflux pumps to actively transport triclosan out of the cell while others depend on certain
enzymes for survival (Welsch & Gillock 2011). The bacterial fatty acid system (FAS II)
synthesizes fatty acids for lipids in the cell membrane. The final rate limiting step in FAS II is
catalyzed by an enoyl-acyl carrier protein (ACP) reductase. Enoyl-ACP reductase exists in three
isoforms: FabI, FabK, and FabL. Most bacteria contain FabI (Zheng et al 2006). Triclosan
inhibits FabI, interrupting the FAS II pathway and making most cells vulnerable to triclosan.
However, other enoyl-ACP isoforms, FabK or FabL, are resistant to triclosan and enable cell
survival. A third mechanism of triclosan resistance is the expression of a triclosan degrading
enzyme, allowing certain microorganisms to utilize triclosan as a carbon source (Welsch &
Gillock 2011).
1.5. Microorganisms
Soils are highly diverse in microbial content. Delmont (2011) writes, “One gram of soil
has been reported to contain up to 10 billion microorganisms and thousands of different species.”
Pepper et al. (2015) explain that one gram of soil contains between 108 and 10
10 bacteria, and
could contain 104 different species of microorganisms. Birds introduce additional bacteria, such
as E. coli, to native bacteria on green roofs through defecation. Collections of fecal coliforms and
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other pathogenic bacteria introduced to green roofs could pose health risks to the general public
(National Water Program 2008). Additional coliforms found in fecal matter and in soil include
genera of Citrobacter, Klebsiella, and Enterobacter (Fiello et al. 2014).
1.6. Biolog EcoPlates™
Biolog EcoPlates™ (Biolog, Hayward, CA) are used to measure microbial community
diversity by analyzing organisms’ ability to degrade certain substances. Each Biolog plate
contains 31 different carbon sources in triplicate. Cell growth is indicated by color change, which
can be read on any microplate reader (Biolog, http://www.biolog.com/products-
static/microbial_community_overview.php).
1.7. Soil DNA Extraction
Analyzing the soil metagenome is challenging because the actual DNA recovered from the
soil is not an accurate representation of all DNA existing in the soil. Though several methods
exist for extracting DNA from soil, none succeed at successfully representing the complete
diversity of soil microorganisms.
There are two types of soil DNA extractions, discussed by Robe (et al. 2003): direct
extraction of nucleic acids through in situ cell lysis and indirect extraction involving first
separating the bacteria from soil followed by cell lysis. Direct lysis yields greater amounts of
DNA and is best for examining the entire diversity of microbes in a given soil sample. After
breaking cell walls, nucleic acids are separated from the soil. Cell lysis methods include physical
methods, such as bead beating or freeze-boiling, chemical methods involving detergents, and the
disruption of cell enzymes. As nucleic acids are purified, their incomplete removal from humic
compounds often results in DNA loss.
Indirect cell lysis requires centrifugation to separate the bacteria from soil. Extracted cells
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are then lysed and the DNA is extracted. Chemicals, blenders, and vortexing are all methods by
which soil can be separated. After centrifugation, the supernatant contains both DNA and humic
compounds. DNA is then purified following the direct lysis method (Robe et al. 2003).
Limited research has been conducted on DNA extractions from recycled rubber green
roof media, and the effects of greywater containing triclosan on such media has yet to be
investigated. Bacteria, whether initially present in green roof media or introduced through
defecation, hold the potential to become resistant to antibiotics after coming into contact with
triclosan (Welsch & Gillock 201). This research examines the use of recycled rubber as an
alternative extensive green roof medium and the effects of triclosan on native microbial
communities.
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Chapter 2: Materials and Methods
2.1 Pots, Media, and Planting
Eight carrying trays of eighteen standard cell inserts (pots) were filled with green roof
media. Trays 1-4 contained recycled media, shredded and crumb rubber with compost, while
trays 5-8 contained commercial media, expanded shale with compost. Individual pots were filled
2 in. deep with media and were planted with landscaper’s sun and shade grass seed, a mixture of
ryegrass and Kentucky bluegrass. Pots were shelved at room temperature beneath grow lights on
a timer that was synchronized to the time of sunrise and sunset for each day simulating the
normal period of daily sunlight.
2.2 Watering
On Mondays, each tray of pots was watered with 500 mL synthetic greywater. Synthetic
greywater contained a mixture of shampoo, laundry detergent, cooking oil, and ethanol (Harrow
et al. 2011). Trays 3, 4, 7, and 8 were watered with greywater containing triclosan dissolved in
ethanol. On Wednesdays and Fridays, each tray was watered with 500 mL tap water. For
consistent sprinkling, water was poured from a graduated cylinder through the perforated cap of
a plastic Buchner funnel. The pot inserts were allowed to drain before placing them back into the
carrying trays. Draining prevented stagnant water accumulation and excess water availability to
the root systems.
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Table 1: Composition of Synthetic Greywater (Harrow et al. 2011)
Component Description Greywater (GW)
(per L tap water)
Greywater plus
Triclosan (GWT)
(per L tap water)
Shampoo
Johnson’s Baby
Shampoo; Johnson
and Johnson
0.80 mL 0.80 mL
Laundry Detergent
Seventh Generation:
free and clear of
perfumes and dyes
0.064 mL 0.064 mL
Cooking Oil Crisco All Natural
Pure Vegetable Oil 0.01 mL 0.01 mL
Triclosan
(CAS 9012-63-9) Sigma 72779
10 mL ethanol with
no Triclosan
20 mg Triclosan
dissolved in 10 mL
ethanol
2.3 Biolog EcoPlate™ Analysis
Biolog EcoPlates™ were used for a physiological diversity analysis. Approximately 2 g
media were added to a centrifuge tube containing 20 mL sterile 1% TSB, which was vortexed for
1 min. After 30-60 seconds, after the particles had settled, the supernatant (a 10-1
dilution) was
used to prepare 10-3
, 10-4
, and 10-5
Biolog dilutions. Separate Biolog EcoPlates™ were
inoculated with 100 µL of each dilution. The plates were incubated at 25°C for 48 hours.
Additional Biolog EcoPlates™ were prepared by inoculating the dilutions with the same
triclosan concentrations as used in the irrigation water. Biolog plates were read using a
Molecular Devices (Sunnyvale, CA) SpectraMax M2 microplate reader.
2.4 DNA Extraction
Soil sampling occurred monthly. The grass was removed from one pot in each of the
eight trays. Soil was aseptically transferred into bead tubes for DNA extraction following the
MoBio PowerSoil® DNA Isolation Kit (MoBio, Carlsbad, CA). Extracted DNA was stored in
microcentrifuge tubes at -20°C.
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2.5 DNA Quantification
To ensure the presence of extracted DNA, the Invitrogen Quant-iT™ broad range dsDNA
Assay Kit (Grand Island, NY) procedure was followed. Fluorescence readings were taken on a
Molecular Devices SpectraMax M2 microplate reader with an excitation setting of 480 nm and
an emission setting of 530 nm.
2.6 Pyrosequencing
Frozen DNA samples representing early media sampling and a later sampling, three
months later, were sent to Research and Testing Laboratory (Lubbock, TX) for 16S
pyrosequencing analysis.
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Chapter 3: Results and Discussion
Vegetation successfully grew in recycled rubber and commercial media (Figure
2), confirming that recycled rubber supplemented with compost is a viable lightweight, artificial
growth medium for green roofs. After initial planting, the grass was slower to grow in the
recycled rubber medium. Once established, grass in the recycled medium grew similarly to the
grass in the commercial medium.
Figure 2: Growth Trays. Recycled rubber (left) and commercial (right) media.
The Shannon diversity index[H = − ∑ (𝑝𝑖)(ln 𝑝𝑖)Si=1 ] was used to measure ecological
diversity in each Biolog plate (Figure 3), illustrating the samples’ – physiological diversity (H’).
Samples rich in microorganisms contain high numbers of different taxa and are more diverse.
The early heterotroph Biolog plates showed significant differences between the recycled GWT
and both the commercial GW and GWT samples. Significant differences between the recycled
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GWT and commercial GWT suggest that the medium, independent of triclosan, has some effect
on microbial physiological diversity. The late heterotroph Biolog plates show no significant
differences between all four sample types, congruent with previous research (Mehalik 2013) and
suggesting that microbial diversity converges on a stable equilibrium community.
Figure 3: Shannon Diversity Indices of Biolog EcoPlates™
The early heterotrophs in triclosan Biolog plates show a significant difference between
the recycled GWT and commercial GW samples. Medium differences, irrigation with triclosan,
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or some combination between the two might be responsible for the physiological diversity, but
no definite conclusions can be drawn from this research.
Biolog plates are useful in analyzing functional diversity among microorganisms.
Depending on organisms’ metabolic similarities and differences, several genomically different
microorganisms might be capable of metabolizing the same nutrients, producing similar Biolog
results. Functional differences highlighted by Biolog plates cannot exclusively determine the
overall microbial diversity. For a more detailed understanding of microbial systems, Widmer (et
al. 2001) suggests combining techniques, such as pairing Biolog data with DNA analyses.
Combining functional diversity with genomic diversity provides a greater summary of overall
microbial diversity.
In previous research (Mehalik 2013), the MoBio PowerSoil® kit was successful at
extracting bacterial DNA from both RM and CM samples. The kit was again successful,
allowing for PCR and pyrosequencing through the Testing and Research Laboratory. The
genomic diversity was analyzed by selecting the most prevalent classes of bacteria in each
medium. Sequencing results categorized as “no hit” were not included in the analysis. Krona
charts (Figures 4-11) provide a metagenomic visualization of the media samples (Ondov et al.
2011).
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Figure 4: Early Recycled GW
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Figure 5: Late Recycled GW
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Figure 6: Early Recycled GWT
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Figure 7: Late Recycled GWT
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Figure 8: Early Commercial GW
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Figure 9: Late Commercial GW
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Figure 10: Early Commercial GWT
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Figure 11: Late Commercial GWT
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In commercial samples, triclosan led to a decrease in Alphaproteobacteria and an
increase in Gammaproteobacteria. Recycled samples showed an opposite decrease in
Gammaproteobacteria and increase in Alphaproteobacteria. Gammaproteobacteria,
gram-negative bacteria, including members of the genus Pseudomonas, Alcaligenes, Aeromonas
and Vibrio, as well as the Enterobacteriaceae, are common environmental isolates with broad
metabolic capabilities.
Members of the Alcaligenaceae family, known degraders of triclosan (Welsch & Gillock
2011), were only present in the recycled medium and showed a hundredfold increase over the
three-month growth period. Pseudomonas, also known to degrade triclosan, decreased in the
recycled medium, but increased in the commercial medium. Previous research showed
Pseudomonas decreased in both recycled and commercial media irrigated with synthetic
rainwater (Mehalik 2013). Commercial aggregates are more porous than rubber, providing a
greater surface area, and might have provided Pseudomonas with increased triclosan access,
accounting for differences in survival.
Near the end of the sampling period, bacterial classes appear to have converged with
similar quantities of taxa present in both types of media. Overall, triclosan appeared to have little
effect on both physiological and genomic microbial diversity.
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Chapter 4: Conclusion
This research confirms that recycled rubber supplemented with compost is a viable green
roof growth medium. The artificial recycled rubber growth medium performs similarly to
commercially available media, and microbial communities in both recycled and commercial
media appear to converge over time. Greywater containing triclosan does not drastically alter
microbial diversity in the short term, but long term exposure to triclosan should be further
investigated. Future research could include monitoring microbial diversity over an extended
amount of time, examining increased accumulations of triclosan within the growth media, and
the bioavailability of triclosan due to medium composition.
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