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Distribution and Abundance of Ciliates in a Green Roof Demonstration

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004089/00001

Material Information

Title: Distribution and Abundance of Ciliates in a Green Roof Demonstration
Physical Description: Book
Language: English
Creator: Fakhri, Mustafa
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2009
Publication Date: 2009

Subjects

Subjects / Keywords: Microbe
Microorganism
Protozoa
Protist
Ciliate
Green Roof
Roof Garden
Soil
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Protozoa are an active part of the soil ecosystem. Their most recognized role in the soil is that of making nitrogen (N) more available to plants through feeding and excretory activities. Soil protozoology is a relatively new field and there are still many environments that have not been investigated. In this study I characterize protozoan populations in a green roof garden demonstration to see how the abundance and diversity of protozoa relates to changes in nitrogen, temperature, and plant growth over time. In addition to relating the populations of large ciliates to particular environmental factors, distribution in relation to spatial scale is analyzed. The five most common genera were chosen for analysis: Colpoda, Oxytricha, Spathidium, Litonotus, and Euplotes. I hypothesized that Colpoda would be the dominant genus and that total abundance patterns would vary over the plot due to differences in microconditions. The first part was verified, as Colpoda turned out to be one of the two dominant genera. Surprisingly, abundance patterns did not vary over the plot. This was attributed to conditions relevant to protozoan life, such as water content, being constant over the entire plot.
Statement of Responsibility: by Mustafa Fakhri
Thesis: Thesis (B.A.) -- New College of Florida, 2009
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Gilchrist, Sandra

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2009 F1
System ID: NCFE004089:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004089/00001

Material Information

Title: Distribution and Abundance of Ciliates in a Green Roof Demonstration
Physical Description: Book
Language: English
Creator: Fakhri, Mustafa
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2009
Publication Date: 2009

Subjects

Subjects / Keywords: Microbe
Microorganism
Protozoa
Protist
Ciliate
Green Roof
Roof Garden
Soil
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Protozoa are an active part of the soil ecosystem. Their most recognized role in the soil is that of making nitrogen (N) more available to plants through feeding and excretory activities. Soil protozoology is a relatively new field and there are still many environments that have not been investigated. In this study I characterize protozoan populations in a green roof garden demonstration to see how the abundance and diversity of protozoa relates to changes in nitrogen, temperature, and plant growth over time. In addition to relating the populations of large ciliates to particular environmental factors, distribution in relation to spatial scale is analyzed. The five most common genera were chosen for analysis: Colpoda, Oxytricha, Spathidium, Litonotus, and Euplotes. I hypothesized that Colpoda would be the dominant genus and that total abundance patterns would vary over the plot due to differences in microconditions. The first part was verified, as Colpoda turned out to be one of the two dominant genera. Surprisingly, abundance patterns did not vary over the plot. This was attributed to conditions relevant to protozoan life, such as water content, being constant over the entire plot.
Statement of Responsibility: by Mustafa Fakhri
Thesis: Thesis (B.A.) -- New College of Florida, 2009
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Gilchrist, Sandra

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2009 F1
System ID: NCFE004089:00001


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DISTRIBUTION AND ABUNDA NCE OF CILIATES IN A GRE EN ROOF DEMONSTRATION BY MUSTAFA FAKHRI A Thesis Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Sandra Gilchrist Sarasota, Florida April, 2009

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Preface I am interested in microbes because they are so ubiquitous, yet there is still so much to learn about them. Getting my first microscope in fifth grade opened up a new world, a world that I could explore freely w ithout even leaving my own backyard. After looking at things such as fly legs and salt crystals, I made a hay infusion with grass from the yard to see if I could find living organism s. It was astonishing to see the life that arose from material that seemed so inert. Watching these critters was entertaining. After viewing a drop of pondwater, I was even more hooked, as the number of organisms I could find seemed limited only by the time spent searching for them. I wanted to explore new territory. Prot ozoa were always described as living in either freshwater or marine habitats, but I wondered about where else they might be. It was amazing to me that I could grow protozoa from the grass in my lawn. It seemed that these organisms could be everywhere, which it turns out they are! The organisms in the hay infusion probably came from cysts on the so il, or even from cysts in the air that happened to fall into the bacteria-rich culture. In my first year at the New College of Florida, I wanted to do an independent study project mapping protozoan populations over campus to see how they varied over different environments. This turned out to be more complicated than it had initially seemed, so instead I took samples from a few selected habitats to see if environmental factors could be linked to pr otozoan diversity and abundance. No strong connections were made. There were so many variables th at it was difficult to discern which factors were responsible for which differences in populations. A more controlled experiment would be needed in which variables could be measured and manipulated. ii

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My project sponsor Dr. Sandra Gilchris t mentioned that some students were setting up a green roof garden demonstra tion, and suggested that it might be an interesting and new microbial habitat to study as well as an environment where variables could be controlled to some degree. The ga rden provided a semi-closed system that was exposed to some environmental factors while also allowing a large degree of control. The soil, wetness, and plants could be contro lled by the experimenter, while the effects of factors like temperature and weather could be observed. It was elevated above the ground by bricks, so the soil outside did not interact directly w ith the soil inside. This setup allowed for the applica tion of island biogeography theory to explain changes in community structure. Also, protozoa on a green roof garden have not been studied before, and this seemed like more new territory to be explored. The study allowed me to investigate something biological, but also see its relevance to practical applic ations. It provided informa tion about island biogeography and protozoan populations in relation to e nvironmental conditions, while also providing information relevant to green roofs and sustai nability. Knowing more about protozoa in these environments will contribute to our unders tanding of the soil, of which protozoa are an integral part. iii

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Acknowledgm ents Thanks to my sponsor Dr. Sandra Gilc hrist, who provided encouragement and vital feedback throughout this process. It also would not have been possible without Lauren Cardella, Jose Villar, and Madison Horn, who lead th e construction of the garden containing the microbes here investigated. iv

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Table of Contents Title Pag e Preface........................................................................................................................ .........ii Acknowledgments..iv Table of Contents................................................................................................................v List of Figures.vi Abstract..........................................................................................................vii Introduction..1 Methods..15 Data Analysis.20 Distribution Nitrogen.29 Temperature...........29 Garden Discussion..33 Island Biogeography..33 Soil Crust...35 Composition...37 Distribution....38 Nitrogen. Rhizosphere References..........42 v

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List of Figures Figure 1 2 Figure 2 3 Figure 3 6 Figure 4 7 Figure 5 7 Figure 6 8 Figure 7 9 Figure 8 15 Figure 9 16 Figure 10 16 Figure 11 17 Figure 12 17 Figure 13 19 Figure 14 18 Figure 15 21 Figure 16 22 Figure 17 24 Figure 18 24 Figure 19 25 Figure 20 25 Figure 21 26 Figure 22 26 Figure 23 27 Figure 24 28 Figure 25 28 Figure 26 28 Figure 27 30 Figure 28 31 Figure 29 32 Figure 30 35 vi

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vii DISTRIBUTION AND ABUN DANCE OF CILIATES IN A GREEN ROOF DEMONSTRATION Mustafa Fakhri New College of Florida, 2009 ABSTRACT Protozoa are an active part of the soil ecosystem. Their most recognized role in the soil is that of making nitrogen (N) more available to plants through feeding and excretory activities. Soil protozoology is a relatively new field a nd there are still many environments that have not been investigat ed. In this study I characterize protozoan populations in a green roof ga rden demonstration to see how the abundance and diversity of protozoa relates to change s in nitrogen, temperature, and plant growth over time. In addition to relating the populations of large ci liates to particular environmental factors, distribution in relation to spatial scale is analyzed. The fi ve most common genera were chosen for analysis: Colpoda, Oxytricha, Spathidium, Litonotus, and Euplotes I hypothesized that Colpoda would be the dominant genus and that total abundance patterns would vary over the plot due to diffe rences in microconditions. The first part was verified, as Colpoda turned out to be one of the two dominant genera. Surprisingly, abundance patterns did not vary over the plot. This was attributed to conditions relevant to protozoan life, such as water content, being constant over the entire plot. Sandra Gilchrist, Ph.D. Division of Natural Sciences

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Introductio n Soil is traditionally described by its non-living components such as mineral composition, organic matter, particle si ze, and pH. However, the biological components of soil are also important desc riptors. Protozoa are relevant to agricultural practices for th eir role in nitrogen cyc ling. Knowledge of these biological properties could improve the health of a horticultural or agricultural plot, and at the same time provide green benefits such as sustainability and decreased need for fertilizer. Protozoa are single-celled eukaryotes that move through watery environments by means of pseudopodia, cilia, or flagella. A watery environment for their size can be a very small amount of water. Though they are commonly known for inhabiting large bodies of water such as ponds and o ceans, the thin film of water around soil particles is sufficient for harboring active pr otozoa (figure 1). Th e protozoa typically found in soil are heterotrophic flagellates, testate amoebae, naked amoebae, and ciliates. Large ciliates were chosen fo r the present study for practical as well as biological reasons. They are more exposed to environmental changes than smaller protozoa. Due to their size, ciliates inhab it the larger pore spaces where they receive more fluctuations of temperature, mois ture, and carbon dioxide, compared with smaller protozoa which inhabit tinier spaces between or within particles where they are somewhat protected from changes in environmental conditions (Bamforth 2001). Many ciliates readily encyst and excyst, so they react quickly to changing conditions. Compared to amoebae and flagellates, ciliates are easier to find due to their large size and conspicuous movement. 1

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Figure 1. 1. fungal hyphae 2. actinomycete hyphae 3. bacterial colony 4. bacteria degrading dead organic matter 5. bacteria degrading dead fungal hyphae 6-8. naked amoebae 9. Zoopagales fungus, which paralyzes amoebae and consumes them 10-11. flagellates 12. ciliate Crytolophosis 13. Colpoda 14-16. testate amoebae 17. nematode. (Bamforth 1980) A green roof garden demonstration wa s constructed at the New College of Florida as part of a larger greening effort for the campus. It was meant to encourage people to think about sustainable devel opment on campus and to think creatively about how this could be achieved. Construction of the garden demonstrates that the placement of gardens on the roofs of campus buildings is a very real possibility that requires only readily available resources. Economic considerations are not an issue, since the cost of creating the garden was minimal. The test garden was placed in an open grassy space on the residential side of campus where there is a high degree of foot traffic so that it could receive plenty of light and be seen by many people (figure 2). 2

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Figure 2. Garden located centrally on the residential side of the New College campus. Beyond the simple definition of plants purposely grown on a roof, a green roof garden must meet certain criteria due to its unique location. The weight of the garden is limited by the strength of the r oof, so minimizing weight is important. The green in green roof garden implies that it is environmentally friendly. By its nature, the garden already makes use of the sun's energy and pr otects the building from excessive heating. However, to determine how green a system truly is, the inputs must be taken into account as well. The garden should efficiently utilize nutrients and water, as these are valuab le resources that should not be used wastefully. 3

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The garden must be suitable for the plants, which have their own requirem ents that must be taken care of in conjunction with the physical elements of a green roof garden. According to ECHO (2008) there are four things roots need: air, water, nutrients, and something to keep the sun and wind from drying them out. There are many ways to meet structur al as well as green parameters. Roof gardens are traditionally divided into tw o categories: extensive (figure 3A) and intensive (figure 3B). An extensive garden typically co ntains only one or two types of plants and functions mainly as a ther mal and hydro regulation unit. A green roof can be simple like sod on a Norwegian house (figure 4) or as complex as an entire park, like Millennium Park in Chicago (figure 5). The weight of the garden must always be taken into account. For exam ple, lightweight, expanded polystyrene was used to create the landforms in Millennium Park. Soil is commonly used in highmaintenance rooftop gardens when the roofs can support more weight, but in many cases soil is too heavy. Instead, synthetica lly expanded clay, such as perlite, can be used as planting medium. The depth of the planting medium is greater for an intensive garden, which must be on a roof de signed to handle greater weight. Such a garden might also be opened up to access by the public. Modern green roofs, defined as a sy stem of manufactured layers and planting medium atop a building, are relativel y new. In the US, the first known green roof was built on the Rockefeller Center in New York City, New York between 19331936 (figure 6). However, it is only in the past two decades that green roofs are becoming more common, due to increased environmental awareness and predictions of climate change (Taylor 2007; Wark and Wark 2003). Construction, encouraged by 4

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grants and b uilding incentives to prospectiv e green roof owners, has been mainly in cities, as the benefits of green roof ga rdens are especially relevant to such environments (Taylor 2007). They can re duce the heat island effect, which is increasingly important due to climate change projections, and they can also make use of excess precipitation to reduce flooding and runoff of pollution into nearby bodies of water. 5

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A) B) Figure 3. Side schematic of an A) extensive roof garden and a B) intensive roof garden. SHADE Consulting 2003 6

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Figure 4. Norwegian Folk Museum, Oslo. The sod roof provides insulation in cold weather. Photo by Kjetil Bjrnsrud (2002). Fig ure 5. Millenium Park is a 24.5 acre public pa rk which is also roof garden, covering a 4000 car capacity underground parking lot, railway tracks, and a 1525 seat indoor theatre. Photo by GRHC (2005). 7

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Figure 6. Photo of Rockefeller roof garden water element. http://gothamist.com/2008/05/14/rockefeller_cen.php In any agricultural environment, it is ideal to make the most of the resources available in a sustainable way. This mean s that resources are being used to their maximum potential at any time while also ensuring that they will be available in the future. Protozoa make resources such as nitrogen more available to plants, and a healthy microbial population can ensure that nutrients will be cycled quickly and efficiently over a period of time (figure 7). Th is is one role of pr otozoa that is being elucidated by research, but there is still much to be understood concerning their role in relation to other pa rts of the soil ecosystem. To begin to understa nd their position in this ecosystem, it is important to firs t have some description of the types and abundances of the protozoa that inhabit soil. 8

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Nitrogen in atmosphere (N2) N-fixing bacteria nitrifying bacteria NH4 + NO2 and NO3 -C exudate (sugars and mucila g e ) b acteria NH4 +Carbon in atmosphere (CO2) pr otozoa Fi gure 7. Nitrogen cycle. On the left, the well-known path involving atmospheric nitrogen being fixed and nitrified by bacteria, and on the right, a path involving carbon exudation, protozoan consumption of bacteria, and protozoan excretion of nitrogen. Drawing of tomato plant from http://grandpacliff.com/Plants/Img-Plants/plant-whole-pt.jpg In addition to their position in the food web and their role in nutrient cycling, protozoa have characteristics that make them particularly well suited as biological indicators, such as short generation times and high reproduction rates combined with a high sensitivity to e nvironmental changes (Foissner 1999). Protozoan distribution, unlike th at of larger organisms, follows a pattern of random dispersal. Species are not restricted geographically, but are instead limited only by their ability relative to other species to survive in a pa rticular environment (Finlay et al. 2001). Such ubiquity also means that any data collected can be applied universally, since it is environmental f actors and not geograp hical location that 9

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determ ines protozoan distribu tion. The description of prot ozoa in different conditions will add to the data pool needed to make use of protozoa as the indicators of such conditions themselves. The present investigatio n should shed light on the colonization and succession of protozoa in a newly created island envi ronment, a green roof garden. A rooftop garden on an aboveground building is not in contact with any other soil environment, and the only way for new materials to be a dded is by aerial disper sal or purposefully by the gardener. However, there is an initia l population of protozoa introduced to the garden with the original soil, and the degr ee to which colonization at later stages is responsible for newly observed specie s is difficult to determine. Island biogeography theory relates th e species richness of an undisturbed island to immigration, emigration, and extinction. Colonization of island environments by protozoa can occur quickly due to the easy dispersal of cysts through air and water. Though not all protozoa have the ability to encyst, those found in soil do. Encystment allows individuals to disperse easily as well as remain viable in unsuitable environments. This ability is especially important for soil protozoa, which inhabit environments where conditi ons such as temperature and moisture change often and quickly. Also, their hab itats are more susceptible to changes in solute concentration, since a small change in dissolved substances can make a large difference in such a small volume of water. Most of the soil protozoan population is encysted at any given moment, so when describing populations it is important to distinguish between total and active counts. Adl and Gupta (2006) suggest that for a particular island habitat the total 10

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species composition does not change from m onth to month, but that there are instead cycles of activity and inactivity for each species. Many methods for describing populations detect only active protozoa. Therefore, when applying the concepts of immigration and extinction to these organisms, one must note that what appears to be immigration might only be excystment, and what appears to be extinction might only be encystment. Because protozoa adhere to the e verything is everywhere principle, dispersal ability is not a ma jor factor in determining th e species composition found in particular environments. Instead, dist ribution depends on environmental factors. Such factors include uneven distribution of prey bacteria, interactions between different protozoan groups, interactions be tween protozoa and other organisms, and restricted movement caused by low water content (Vargus and Hattori 1990). Community structure within the garden will change over time. Succession may be caused by an increasing number of species and the resultant changes in interactions, as well as by changes in th e environment like the breakdown of organic matter. Beyond the initial change in protoz oan community structure when the soil is emptied from the bags into the garden, succession includes the increase in microbial activity that occurs as nutrient cycles are established over time. The environment that soil protozoa i nhabit is small in scale, and factors important to protozoan life can vary over short distances. For example, the spatial structure of ciliate food sour ces has been correlated to distances as small as 4 mm (Grundmann and Debouzie 2000 phide Acosta-Mercado 2003). Microscale environments must be taken into account when sampling in order to better connect 11

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variab les to their effects a nd to avoid generalizing over la rge areas when it is not appropriate. Even once samples are in the lab, microenvironments are likely to exist within a sample, or even within a petri dis h, since there are differences at the cm level or smaller (Acosta-Mercado 2003). A high level of microbial activity occurs in the region surrounding plant roots known as the rhizosphere. Plants not only take up nutrients and water through their roots, but release substances as well Bonkowski (2004) found that plants may release up to half of their fixed C to fuel microbial interactions. While this is an estimate for the upper limit of C exudation, more general estimates like 5-21% are still very high (Marschner 1995). This is an extremely significant amount, as this carbon is not going directly to the growth of plant tissue, nor is it being used in metabolic processes. Through exudation, pl ants modify the soil environment around them to better suit their needs, including nitrogen acquisition (figure 7). By releasing carbon into the soil, the plant increases th e abundance of bacteria around its roots. Larger organisms have lower nitrogen to carbon ratios than smaller ones, so when protozoa graze on the bacter ia, they excrete the excess nitrogen into the surrounding soil. This nitrogen is no longer locked up in bacterial biomass and becomes available for absorption by the plant. While the rhizosphere is known in the field of soil microbiology for its high level of activity, the soil environment in th e green roof garden is different, and might not contain a comparable rhizosphere (figure 8). The soil layer is only about 2 cm thick, and the roots probably travel down further than that, so most of the carboncontaining root exudates woul d get washed away. This could decrease protozoan 12

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abundance and alter nut rient cycling. The soil in the green roof demonstrati on garden is unique compared to other garden types in that it might not be fully rhizospheric, but still surrounds a plant. Part of the soil's purpose is to supply the pl ant with nutrients. However, if the roots go past the soil, then they are not there to acquire the nutrients, and exudates are washed away instead of entering the micr obial loop. Not only does the plant lose a large amount of fixed carbon, it also misse s out on the nitrogen that would be provided by the soil protozoa. In reality, some of the roots probably pass through the soil so that there is limited rhizospheric activity, but also a loss of potential nutrient cycling by the root portions which go past th e soil. The total abundance of large soil ciliates is expected to increase over time in garden systems. This is especially true if the soil was packaged and so its activity was initially low. The population will reach a peak and then remain near a certain elevated level, since food webs will beco me established and nutrients will be continuously cycled through these systems. My initial predictions were that there would be a large variation in abundance, community structure, and the timing of such changes between different sampling squares due to differences in conditions at the micro-scale. Abundance was expected to increase more during warmer weather and less during colder times, since cellular activity is greater at higher temperatures. Even though the roots were expected to penetrate past the soil layer, some of the roots would probably be in the soil, so rhizospheric activity was still expected. N itrogen content over the entire plot was likely to increase due to its mineralization by the protozoa, but was expected to be 13

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14 even higher in areas of greater plant density due to higher levels of microbial activity and nutrient cycling. Protozoan abundance wa s also expected to be higher in these squares due to the greater bacteria l abundance supported by root exudation. Community structure was expected to vary due to differences in conditions at the micro-scale. Colpoda would be the dominant genus as is typically found for most soils due to its ability to tolerate a wide range of conditions and to encyst and excyst rapidly.

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Methods A green roof demonstration was cons tructed using a design from ECHO (2008). The garden consisted of a layer of pierced aluminum cans covered with a thin layer of soil about 2-3 cm thick (figure 8). The so il was a 4:1 ratio of Miracle-Gro's Earthgro potting soil and Miracle-Gro's Earthgro manure. A layer of cinder blocks comprised the base of the garden to simulate a rooftop and to separate the garden from the ground underneath. Wooden boards were placed in a rectangle to create a 1.2 m x 2.4 m enclosure with a height of about 15 cm (figur e 9). Cans pierced with a knife until they had about 10 holes each were placed into th e enclosure until they covered the top surface of the bricks (figure 10) S eeds for tomatoes, green beans, peas, cucumbers, zucchinis, cabbages, nasturtiums, watermelons, pumpkins and peppers were planted in small pots with Miracle-Gro's Earthgro potting soil for three weeks before the young plants were transferred to the garden (figure 11) and organized according to 30 cm2 patches (figure 12). The only regular maintenance was the addition of Sarasota County tap water once every rainless day aside from days 36-56. On day 10, compost was added, and on day 11, a trellis was added to the left side, or the A side of the garden, for the pea plants to grow upon. w ooden barrier ans cinder blocks pierc ed aluminum c soil Figure 8. Side schematic of the demonstration roof garden. Cement bricks simulate a rooftop, pierced cans and soil comprise the planting medium. 15

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Figure 9. New College demonstration garden before substrate added. Figure 10. Pierced can substrate of the demonstration roof garden. 16

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Figure 11. Soil covers the cans and plants are added to the garden. A B C D A B CDEF G H sam pling site plant Figure 12. Grid system created for taking samples for analysis. Sampled squares are in bold. 17

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Soil was collected with plastic spoo ns once a week from the top cm of the bed from each of 5 squares: AA, BC, CE, DG, and CH. Samples were taken from 3 sites within each square for a total of 15 samples. The soil was stored in re-sealable bags and preserved by refrigeration until it was processed over the next few days. Four grams of soil were mixed with eight mL of Zephyrhill s spring water in a 50 mL flask to provide enough wetting to encourage activation of the ciliates as well as make wet slide preparation and observation with a light microscope possible. The flask was swirled by hand at a rate of 100 swirls per minute for one minute to sufficiently wet the samples without destroying the protozoa (Stevik 1998). The slurry was placed in the dark for six hours before being examined. Ten drops from each sample were examined under a light microscope at 100x and ciliates larger than 50 micrometers were sketched and counted. Soil temperature was recorded twice a day, at 7 AM for the day's minimum temperature and at 2 PM for the maximum temperature. Ni trogen content was measured once a week using soil test kits from Ferry -Morse Seed Company (f igure 13), which gave readings on a four step scale ra nging from very low to high. Data were collected for 65 days fr om November 9, 2008 to January 13, 2009. This length of time should allow for a reason able understanding of the changes in ciliate populations relative to the ne w growing habitat for the winter months of Florida. 18

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Figure 13. Example of simple soil test kit used for this project. http://www.drgoodearth.com/image/kids_main_02.jpg 19

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Data Analysis The five most abundant large ciliate genera were Colpoda, Oxytricha Litonotus Spathidium and Euplotes (figure 14). The total abundan ce of large ciliates clearly increased over the 65 day period (figure 15). The maximum occurred at the last data collection, so the abundance may have never reached its peak and probably continued increasing. Average abundance incr eased at a rate of 4.2 ciliates g-1 per day. The abundance for each genus was higher at the end than at the beginning. Initial abundances for the five genera were low, between 0 and 25.32 g-1 Final abundances ranged from .52 to 219.2 g -1 Colpoda Oxytricha Litonotus Spathidium Euplotes encysts bacterivore 50-110 um encysts bacterivore 100-150 um encysts predator 120 um encysts predator 110-250 um encysts bacterivore 80-200 um Figure 14. Common large ciliates of the demonstra tion garden. Those described as predators consume other protozoa. Drawings by Jahn and Bovee (1949) 202

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Abundances of the two dom inant genera remained far above those of the other three for the entire sampling period (figure 16). Oxytricha had the highest abundance for the first four weeks, and Colpoda had the highest abundance for the last four weeks. These two genera combined made up 93-98% of the total ciliat e population for any given week. Maximum abundance portions out of the total are 93% for Oxytricha on day 22, and 72% for Colpoda on day 65. 010203040506070 0 50 100 150 200 250 300 350 f(x) = 4.22x + 15.73Large Ciliate Abundance Total per g Linear regression for Total per gTime (days)Abundance (individuals per g)Figure 15. Abundance for all samples combined over time. 212

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29162229365865 0 50 100 150 200 250 300 350 Proportions for genera out of total spathidium, litonotus, euplotes colpoda oxytrichaTime (days)Abundance (individuals per g) Fig ure 16. Combined totals for the various genera of large ciliates Abundance patterns varied be tween genera. On day 36, Oxytricha populations decreased, while Colpod a populations continued to increase. Abundance changed more erratically for Oxytrich a than for Colpoda, which had a fairly steadily increasing abundance from day 22 to the end of the experiment (day 65) (figure 17). While Oxytricha abundances decreased by la rge numbers three times, Colpoda only had one large dip in abundance (day 22). On the two days that Colpoda abundance decreased, Oxytricha abundance increased, and on the three days that Oxytricha abundance decreased, Colpoda abundances increased. It may be that an increase in one caused a decrease in the other, though if this is the case, then ther e would be some lag time. This remains a possibility even though the changes occurred on the same days, since the lag time may be small due to the fa st reproduction rate of these organisms and 222

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the data collections m ay have been too far apart to detect the lag time. Individuals of different genera may react differently to environmental changes. Colpoda is known for encysting rapidl y, enabling it to survive dry environments. A drying of the soil would therefore cause more Colpoda to survive than Oxytricha which cannot encyst as readily a nd would be destroyed by the drop in moisture. The abundances for both genera might affect one another. If they occupy a similar niche, then a decrease in abundan ce of one genus might allow members of the other genera to occupy more of that space. Oxytricha and Colpoda both consume bacteria, so it is possible that a decrease in Oxytricha abundance would increase food abundance for Colpoda, and vice versa. Abundances for the other three genera, Litonotus Spathidium and Euplotes also followed different patterns (figure 18). There was only one day on which all five genera showed the same trend, which was on day 22 when all genera increased in abundance. The change in abundance changed similarly on certain days for all genera (figures 19 and 20). On day 29, change in abunda nce increased for all genera except Oxytricha and on day 36 the change in abundance for all genera decreased. There may have been environmental conditions on these days that affected all genera equally. 232

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010203040506070 0 50 100 150 200 250 Abundance of Oxytricha and Colpoda oxytricha colpodaTime (days)Abundance (individuals per g)Figure 17. Abundance for the two most common genera. 010203040506070 0 2 4 6 8 10 12 14 Abundance of Spathidium, Litonotus, and Euplotes spathidium litonotus euplotesTime (days)Abundance (individuals per g)Fi gure 18. Abundance for the three less common genera. 242

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010203040506070 -80 -60 -40 -20 0 20 40 60 80 Change in Abundance of Oxytricha and Colpoda oxytricha colpodaTime (day)Abundance (individuals per g) Figure 19. Abundance change between sampling days. Rate adjusted for larger gaps, such as day 36 to 59. 010203040506070 -4 -3 -2 -1 0 1 2 3 4 5 Change in Abundance of Spathidium, Litonotus, and Euplotes spathidium litonotus euplotesTime (day)Abundance (individuals per g)Fi gure 20. Abundance change between sampling days. Rate adjusted for larger gaps (day 36-59). 252

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100 m Figure 21. Oxy tricha near a soil particle 400x 100 m Figure 22. C olpoda 200x 262

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Distribu tion Total abundance changed according to the same pattern for a ll squares sampled. There were two dips in the first four week s at day 9 and 22, but after that, all squares except DG (which had one dip) had only in creasing abundances (figure 23). Final abundances by square were spr ead from 112 to 486 ciliates g-1. The square with the highest plant de nsity, CE, had the lowest abundance of ciliates. Abundances for the squares showed no trend with plant density. 010203040506070 0 100 200 300 400 500 600 Abundance in squares over 65 days AA BC CE DG CHTime (days)Abundance (individuals per g)Fi gure 23. Abundances in sampling squares over time. 272

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Square Plant Density AA 2 BC 0 CE 4 DG 2 CH 0 Figure 24. Plant density in the squares sampled. No te that because the plants differed, the root systems and the aboveground biomass also differed. Square Plant density for square plus surrounding squares AA 6 BC 4 CE 18 DG 10 CH 7 Figure 25. There were differing nu mbers of plants around the squares being sampled. These differences could have contributed to fluctuations over time for the microbes, serving as both sources and sinks. DayNitrogen level at BBNitrogen level at CE 203 913 1610 2200 3620 5823Figure 26. Gross levels of nitrogen for two different squares. (Numbers for nitrogen units are derived from a scale where 0=very low, 1=low, 2= medium, and 3=high) 282

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Nitrogen Nitrogen co ntent was measured for CE because it has a high plant density, and from BB because it has a low plant dens ity. This type of measurement is only a crude determination of variation. The data are insufficient for a proper trend analysis, though they do demonstrate that nitrogen levels varied over the plot and over time. More precise analysis of total nitrogen and availa ble nitrogen is needed to asses this aspect more fully. Even though CE had a high nitrog en content for some weeks, it gave a count of zero for other weeks (figure 26). Temperature The lowest temperature of 5.3oC was recorded on day 11, so its effect was expected to show up in the following samp le collection, which was on day 16. This was in fact one of the two dips in abundance for the total population, so it is likely that the cold temperature slowed down cellular activ ity and reproduction. It may also have resulted in some of the ciliates encysting. Overall, temperature was higher during the second half of the sampling period than the first half (figure 27). The lows were higher during the later half, and the highest temperature of 28.9oC also occurred during this half, on day 57. Higher temperatures may have contributed to the populat ion growth during the second half. 292

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010203040506070 0 5 10 15 20 25 30 35 Daily Soil Temperature High LowTime (day)Temperature (C) Figure 27. Range of soil temperature. 303

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Garden The plants in the garden appeared healt hy at the end of the 65 day period (figure 29). There were flowers as we ll as vegetables such as gree n beans and peppers. It was expected that the plants woul d show signs of nutrient deficiency over time, as nutrients might get used up or washed away from the thin layer of soil. The results showed otherwise, which is a plus for sustainability since the plants were able to mature enough to provide vegetables without the need for fertilizer, and with only a minimal amount of soil. Figure 28. Garden at day 60. 313

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A B Figure 29. A. Long view of garden at end of project. B. Fruiting (green beans). 323

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Discussion Island Biogeography A green roof garden is an island, in that it is an en vironment suitable to certain organisms surrounded by an environment uns uitable to those organisms. The soil initially contains a population of microbes, even though they might only be encysted forms. These organisms were transported to the garden along with their previous soil materials, though the environment is different in the new location. A green roof garden is exposed to high levels of sunlight and wind, a ffecting both temperatur e and water. It is also not near other soil environments. The demonstration garden was placed in a sunny area with no tree cover to simulate a rooftop climate. Unlike an actual rooftop garden, the gard en in this project was surrounded by soil. However, it was stil l an island in that the surrounding soil was different, as it was sandier and contained le ss organic matter than the garden soil. In a newly created, relatively sterile island, colonization by protozoa can occur quickly because of the easy di spersal of cysts through the ai r and water. Protozoa can persist in the environment surrounding an island due to their encystment capability. Even within the soil, most of the protozoan populatio n is encysted at any given time. Visual cyst identification is difficult for all but a few more conspicuous va rieties. While new molecular techniques may enable researchers to identify initial populations more fully (Dunthorn et al. 2008), with traditional methods only the active forms for that given moment can be counted, repr esenting only a small portion of the total population. Opportunistic species that are successful in a given environment but only excyst for certain periods of time may go undetected in prot ozoan counts. It is difficult to determine 333

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whether a sp ecies is newly arri ved or freshly excysted, or if a species has disappeared or whether it is just encysted for the time. Detecting migration and extinction for protozoa therefore requires specia lized methods compared with those for larger organisms. In the present study, it is the changes in populations of active protozoa that are measured, not the changes in total population. It is likely that most of the genera enumerated were already present in the initial soil. Indeed, on the firs t day, three of the five genera we re present in detectable numbers (figures 17 and 18). For these genera, reproduction, not colo nization, resulted in increasing abundance over time. The other tw o genera appeared by day 22, so even if they were new colonizers, they colonized a nd reproduced to detectable numbers quickly. However, as all five genera have encystment capabilities, it is likely that these two were also already present in the soil. Finlay and colleagues (2001) found 203 species after manipulating temperature, light, and food sources in a freshwater sediment culture that initially yielded 20 species. Foissner (1997 phide Bamforth 2001) also found that culturing of a soil sample over several years periodically revealed new species. For the green roof garden, the change in conditions when the soil was transferred from the storage bag to the garden resulted in a di fferent population and community structure. Individuals of some protozoa genera probably excysted, while others that were already active increased in abundance. 343

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Soil Crust Figure 30. Schematic diagram of a biological soil crust with lichen, bryophyte, and cyanobacterium colonizers. Belnap and Lange (2002). In many ways, the thin layer of soil in a green roof garden is similar to the soil crusts found in nature. A crust is the top few millimeters of soil, consisting of lichens, bacteria, protozoa, nematodes, and microfungi which act to hold together soil aggregates and create an environment distinct from that of the soil below. Bamforth (2008) did a study on such crusts in a c ool desert and found that Colpoda dominated, similar to the results of the green roof demonstration st udy. Though the crust is not rhizospheric, it contains nitrogen fixing organi sms such as cyanobacteria (Lange and Belnap 2002) as well as nitrogen mineralizing protozoa. Sim ilarly, the soil in the green roof garden 353

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dem onstration might not be rhizos pheric, but it may still play a role in providing nitrogen to plants. Crusts typically develop, rather than diminish, over time (Garcia-Pichel et al. 2003). Studies on crusts suggest that si milar environments like the soil in the demonstration garden may be sustainable. The crust ecosystem provides a good example of organisms that should be placed in a gr een roof garden system where the amount of soil is limited. Traditional agricultural and horticultural methods destroy biological crusts (Johnson et al. 2007) but if such an environment could be maintained by thoughtful gardening, a sustainabl e nitrogen-cycling system could be maintained for long periods with a minimal amount of soil. The biomass within crusts is often higher th an that of deeper soil, and even within a crust, the uppermost layer has the highest mi crobial activity (Garcia-Pichel et al. 2003). In the green roof demonstration study, the top cm the thin soil layer was investigated, and increasing protozoan abundance over time was found. Nagy and colleagues (2005) studied prokar yotic distribution in crusts of the Sonoran desert and found no difference in diversity and composition between sites under plant cover and those which were not. Though the study was conducted for prokaryotes, its results may be indicative of microbial lif e in general. The pattern of increasing abundance and lack of variation over the plot in the green roof garden demonstration suggests a similar situation in which nutrien t cycles are established independently of plants. 363

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Crusts are extrem e environments in terms of light exposure, temperature fluctuation, physical abrasion, and changes in water availability (Johnson et al. 2007). The soil layer in a green roof garden faces similar extremes such as high exposure to sunlight and wind. The healthy development of the plants in the demonstration garden with only a small amount of non-rhizospheric so il suggests that nutrient cycles similar to those in soil crusts were estab lished, and indicates that a prac tical application of soil crust biology to green roof gardens could improve th e efficiency and sustainability of such systems. Composition The wetting of samples before viewing undoubtedly aff ects the population composition. One way it does this is by affecti ng excystment. It has been widely held that drying causes protozoa to encyst. However, there is no known mechanism in ciliates for direct detection of drying. Ekelund and colleagues (2002) found that addition of water alone did not stimulate excystment in collected soil samples. Though water is required for excystment, it is not a sufficient stimulus by itself. It has been proposed that the ciliates might somehow det ect bacterial abundance, CO2 from other ciliates, or possibly a chemical secreted by other ciliates. Colpoda encystment can be induced by an increase in ion concentrations, though this e ffect can be inhibited in the presence of bacteria (Watoh et al. 2003 phi de Yamasaki et al. 2004). This can be viewed as an indirect mechanism for detection of drying. Wetting of the soil still affects excystment since it alters the immediate environment of th e cell both physically and chemically. In natural soil environments, changes in ion c oncentrations are likely to occur often and 373

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dram atically due to the repeated drying and re -wetting of a small volume of soil. In the lab, the addition of a relativel y large volume of water coul d reduce ion concentrations, causing protozoans like Colpoda to excyst. The method used in the present study favors genera that can excyst within six hours. Since Colpoda can excyst more readily than other ciliates, it is possible that a higher porti on of them were found compared to others, even though in the natural environment thei r activity relative to others might be less dramatically elevated. The total abundance for the five genera showed an increasing trend, and did not show any signs of peaking (figure 15); no da ta for individual species were determined from these samples. There is typically a carrying capacity for protozoa in soil environments (Clarholm 2005), though abundances did not reach this level within the 65 days over which the study was conducted. Colpoda are well-known for thriving in terrest rial habitats. Bamforth (2001) found that in times of environmental stre ss, such as cold weather or drought, Colpoda made up 50-100% of the active ciliate population. In the demonstration garden as well, Colpoda reacted less to the extreme cold, and even when its abundance decreased, the decrease was less than that of the other dominant ciliate, Oxytricha Distribution Total abundance as well as abundance for each genus changed according to the same pattern for all the sampling areas. This appears contrary to the original prediction that the abundance pattern would vary due to different conditions ove r the plot. It is likely that certain conditions did not vary over the plot as much as expected, or 383

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alte rnatively, that the conditions which did vary were not crit ical for these ciliates. The soil was watered equally over the entire plot so if water content is a major factor affecting abundance, then abundances would change equally over the plot, which they did. Though water content did not vary over th e plot, it may have varied over time and affected temporal abundance patterns. The garden was watered regularly, but it is possible that weather conditions such as sunlig ht, temperature, and rain affected the water content of the soil so that it was not actually constant. Th e amount of water in the soil can have a large impact on protozoa migra tion. Vargus and Hattori (1990) found that Colpoda were unable to migrate from one pore to another at 60% water-holding capacity (WHS), but were able to migrate at 80% WHS. Also, rain can cause sudden dispersion of cells by suspending them in moving water. Sunlight, on the other hand, can cause evaporation, decreasing water co ntent and reducing migration. Water has relevant effects beyond protozoa n motility and cellular function. It allows for bacterial growth, so the constant addition of water to the garden would have caused bacterial abundance, and that of thei r protozoan grazers, to remain high and possibly equal over the entire plot. Nitrogen Nitrogen content was intended to be a measure of protozoan activity rather than a factor affecting it. Higher protozoan a bundances have been found to cause nitrogen levels to increase. Areas with higher plant density are expected to have higher microbial populations, and therefore more nitrogen mineralizing protozoa (Bonkowski 2004). Nitrogen varied between the tw o squares tested, but no clear pattern emerged. Collection 393

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of nitrogen data for m ore points in the garden would be helpful. The dramatic changes in nitrogen content, like the swings from high to undetectable levels in just a week, could mean that nitrogen content changes quickly over time. However, it could also mean that nitrogen content varies over small distances so that the soil co llected had conditions separate from those of the soil just a few mm or cm away. The soil collected at each sampling is assumed to be the same soil from we ek to week, but in r eality it is a different soil that neighbors the soil colle cted the previous week. When sampling with such a method, it may be difficult to separate the effects of spatial versus temporal variations. Rhizosphere The lack of correlation between plan t density and abundance suggests that a rhizosphere does not exist in this type of roof garden configuration. It is surprising that the square with the highest plant density had the lowest abundance, since this suggests that the plants are inhibiting rather than stimulating the pr otozoan population. To better assess the rhizospheric properties of the garde n, it would be helpful to measure the root density in the soil. To determine possible lo ss of rhizospheric activit y, it would be useful to also determine the portion of root biomass that goes past the soil. Analysis of large ciliate abundance fo r the green roof demonstration garden shows that a sizable protozoan populati on can exist in a small amount of nonrhizospheric soil. The success of the plants demonstrates that the soil was able to provide enough nutrients without the need for fertil izer. Though sustainability beyond the 65 days remains unknown, the soil can be describe d as healthy in terms of its ability to support plants for at least tw o months, or sufficient for at least one growth cycle of 404

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seedling to m aturity. The garden also met green standards by being efficient in using both natural and economic resour ces. Though more re search is needed to better connect the plant growth in such a garden with th e microbial population, this study provides an example of the abundance patterns for certain protozoa over time and space, as well as suggesting that the microbial population was strong enough to provide the plants with what they needed. 414

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References Acosta-Mercado, D., and Lynn, D. H. (2003). The edaphic quantitative protargol stain: A sam pling protocol for assessing soil ciliate abundance and diversity. Journal of Microbiological Methods, 53 (3), 365-375. Adl, M. S., and Gupta V.V.S.R. (2006) Protists in soil ecology and forest nutrient cycling. Canadian Journal of Forest Research, 36(7), 1805-1817 Bamforth, S. S. (2001). Proportions of active ciliate taxa in soils. Biology and Fertility of Soils, 33(3), 197. Bamforth, S.S. (2008). Protoz oa of biological soil crusts of a cool desert in Utah. Journal of Arid Environments, 72, 722. Bonkowski, M. (2004). Protozoa and plant gr owth: The microbial loop in soil revisited. The New Phytologist, 162 (3), 617. Clarholm, M. (2005). Soil protozoa: An under-researched microbial group gaining momentum. Soil Biology Biochemistry, 37 (5), 811. Dunthorn, M., Foissner, W. and Katz, L. ( 2008). Molecular phylogenetic analysis of class Colpodea (phylum Ciliophora) usi ng broad taxon sampling. Molecular Phylogenetics and Evolution, 46, 316. ECHO (Educational Concerns for Hunger Orga nization). (2008). Rooftop and Urban Gardening. http://www.echotech.org. 424

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Ekelund, F., Frederiksen, H. B., and Ronn, R. (2002). Population dynam ics of active and total ciliate populations in arab le soil amended with wheat. Applied and Environmental Microbiology, 68( 3 ), 1096. Finlay, B. J., Esteban, G. F., Clarke, K. J., and Olmo J. L. (2001). Biodiversity of terrestrial protozoa appears homogeneous across local and globa l spatial scales. Protist, 152(4), 355. Foissner, W. (1997). Global soil ciliate (P rotozoa, Ciliophora) diversity: a probabilitybased approach using large sample co llections from Africa, Australia, and Antarctica. Biodiversity Conservation, 6, 1627-1638. Foissner, W. (1999). Soil prot ozoa as bioindicators: Pros and cons, methods, diversity, representative examples. Agriculture, Ecosystems & Environment, 74 (1-3), 95-112. Garcia-Pichel, F., Johnson, S. L., Youngkin, D ., and Belnap, J. (2003). Small-Scale Vertical Distribution of Bacterial Bioma ss and Diversity in Biological Soil Crusts form Arid Lands in the Colorado Plateau. Microbial Ecology, 46, 312-321. Johnson, S. L., Neuer, S., and Garcia-Piche l, F. (2007). Export of nitrogenous compounds due to incomplete cycling within biological soil crusts of arid lands. Environmental Microbiology, 9(3), 680-689. 434

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Lange O.L., and Belnap J. 2002. Biologica l Soil Crusts: Struct ure, Function, and Managem ent. Springer. Marschner, H. (1995). Mineral Nutrition of Higher Plants, Ed 2. Academic Press, London. Nagy, M. L., Perez, A., and Garcia-Pichel, F. (2005). The prokaryotic diversity of biological soil crusts in the Sonoran Desert. FEMS Microbiology Ecology, 54, 233245. Stevik, T. K., Hansenn, J. F., and Jensenn, P. D. (1998). A comparison between DAPI direct count (DDC) and mo st probable number (MPN) to quantify protozoa in infiltration systems. Journal of Microbiol ogical Methods, 33(1), 13. Taylor, D. A. (2007). Growing Green Roofs, City by City. Environmental Health Perspectives. 115(6), A306-A311. Vargus, R., and Hattori, T. (1990). THE DISTRIBUTION OF PROTOZOA AMONG SOIL AGGREGATES. FEMS Microbiology Ecology, 74 (1), 73. Wark C.G., and Wark W.W. (2003). Green Roof Specifications and Standards. The Construction Specifier 56(8) Watoh, T., Yamaoka, M., Nagao, M., Oginuma, K., and Matsuoka, T. (2003). Inducing factors for encystment and excystment in Colpoda sp. Japanese Journal of Protozoology, 36, 105-111 (in Japanese). 444

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454 Yamasaki, C., Kida, A., Akematsu T., and Ma tsuoka, T. (2004). Effect of components released from bacteria on ency stment in ciliated protozoan Colpoda sp. Japanese Journal of Protozoology, 37(2), 111-117.


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