i

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

ii

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

iii

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.

iv

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

vi

List of Tables

Table 1: Composition of Synthetic Greywater ............................................................................... 8

vii

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.

1

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

2

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

3

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

4

(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

5

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

6

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.

7

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.

8

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.

9

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.

10

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

11

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,

12

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).

13

Figure 4: Early Recycled GW

14

Figure 5: Late Recycled GW

15

Figure 6: Early Recycled GWT

16

Figure 7: Late Recycled GWT

17

Figure 8: Early Commercial GW

18

Figure 9: Late Commercial GW

19

Figure 10: Early Commercial GWT

20

Figure 11: Late Commercial GWT

21

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.

22

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.

23

References

Alliance for the Prudent Use of Antibiotics (APUA). (2011). Triclosan. White Paper prepared by

the Alliance for the Prudent Use of Antibiotics. Online:

http://www.tufts.edu/med/apua/consumers/personal_home_21_4240495089.pdf.

Accessed November 21, 2014.

Bridgestone. (2007). Real Answers. Online:

www.bridgestonetrucktires.com/us_eng/real/magazines/bestof3/speced3_tire_component

s.asp. Accessed December 16, 2014

Fiello, M., Mikell Jr, A. T., Moore, M. T., & Cooper, C. M. (2014). Variability in the

characterization of total coliforms, fecal coliforms and escherichia coli in recreational

water supplies of north mississippi, USA. Bulletin of Environmental Contamination and

Toxicology, 93(2), 133-137. doi:10.1007/s00128-014-1299-1

Harrow, D.I., Felker, J.M., Baker, K.H. (2011). “Impacts of Triclosan in Greywater on Soil

Microorganisms,” Applied and Environmental Soil Science, vol. 2011, Article ID

646750. doi:10.1155/2011/646750

Mehalik, L.K., (2013). Methods for the genomic characterization of the microbial community in

a newly developed green roof medium. Unpublished.

National Center for Biotechnology Information (NCBI). PubChem Compound Database;

CID=5564, pubchem.ncbi.nlm.nih.gov/compound/triclosan. Accessed December 16,

2014.

Negahban-Azar, M., Sharvelle, S. E., Stromberger, M. E., Olson, C., & Roesner, L. A. (2012).

Fate of graywater constituents after long-term application for landscape irrigation. Water,

Air, & Soil Pollution, 223(8), 4733-4749, doi:10.1007/s11270-012-1229-y

Ondov, B. D., Bergman, N. H., & Phillippy, A. M. (2011). Interactive metagenomic visualization

in a web browser. BMC Bioinformatics, 12(1), 385-385. doi:10.1186/1471-2105-12-385

Pepper, I.L., Gerba, C.P., Gentry, T.J. (2015). Microbial diversity and interactions in natural

ecosystems. Environmental Microbiology, 3rd

ed. doi: 10.1016/B978-0-12-394626-

3.00019-3

Salsedo, C. A. (2011). Greywater. The SAGE Reference Series on Green Society: Toward a

Sustainable Future. Green Technology (pp. 241-244). Thousand Oaks, CA: SAGE

Reference. Retrieved from

http://go.galegroup.com/ps/i.do?id=GALE%7CCX1560500075&v=2.1&u=psucic&it=r&

p=GVRL&sw=w&asid=cede6d097f8ba8a7b7da993c3795a920

24

Solano, S.L., Ristvey, A.G., Lea-Cox, J.D., and Cohan, S.M. (2010). Crumb Rubber as an

Ammendment for Extensive Green Roof Substrates: Preliminary Research Conclusions.

SNA Research Conference Vol. 55.

USEPA, 2003. Urban Nonpoint Source Fact Sheet. Online.

http://water.epa.gov/polwaste/nps/urban_facts.cfm. Accessed December 13, 2014.

USEPA (2010). Scrap Tires: Handbook on Recycling Applications and Management for the U.S.

and Mexico. Online:

http://permanent.access.gpo.gov/gpo12146/ScrapTireHandbook_US-Mexico2010-

LR.pdf. Accessed December 16, 2014.

USEPA (2013). Common Wastes and Materials – Scrap Tires – Civil Engineering Applications.

Online: www.epa.gov/osw/conserve/materials/tires/civil_eng.htm. Accessed December

13, 2014.

Welsch, T. & Gillock, E. (2011). Triclosan-resistant bacteria isolated from feedlot and residential

soils. Journal of Environmental Science and Health, Part A, 46(4), 436.

doi:10.1080/10934529.2011.549407

Widmer, F., Fließbach, A., Laczkó, E., Schulze-Aurich, J., & Zeyer, J. (2001). Assessing soil

biological characteristics: A comparison of bulk soil community DNA-, PLFA-, and

biolog™-analyses. Soil Biology and Biochemistry, 33(7), 1029-1036.

doi:10.1016/S0038-0717(01)00006-2

Zheng, C., Sohn, M., & Kim, W. (2006). Atromentin and leucomelone, the first inhibitors

specific to enoyl-ACP reductase (FabK) of streptococcus pneumoniae. The Journal of

Antibiotics, 59(12), 808-812. doi:10.1038/ja.2006.108