Transect Study Report
General Biology II
April 7, 2019
A bioretention basin is a shallow basin used to slow and treat on-site stormwater runoff. Stormwater runs through the basin and then filters through the pipes of the basin where it is stripped of chemicals and debris. Measuring biodiversity can be used as a method to determine the overall health of an ecosystem. Higher levels of diversity encourage resilience to extreme weather, disease, and changing environmental conditions. This report uses an array of methodologies such as a hay infusion, serial dilution, gram staining, and Berlese Funnel to investigate biodiversity. We found several different protozoa such as Neourostylopsis, Euplotes, and Stylonychia in Niche 1 of the hay infusion. We also found that bacterial colonies in the nutrient agar are smaller in size than the tetracycline plates, then for the gram dying. We also found several invertebrates with a majority of 5 of them being Arthropoda and Insecta in the transect area. This suggests that the bioretention basin is a healthy and vibrant environment with several ecosystems. These species in each ecosystem are all linked to one another through a food chain beginning with primary producers and ending with higher-level consumers.
This ecological investigation explores the species diversity, abundance, and richness at American University’s bioretention basin. A bioretention basin is a depressed area of land filled with vegetation that collects polluted runoff from the nearby habitat to restore aquifers and filter pollutants out of stormwater.
A bioretention basin is typically constructed with a network of perforated pipes at the bottom of the depression covered by gravel, soil, and plants. As polluted water flows through the soil and vegetation, contaminants such as nutrients, chemicals, hydrocarbons(1), and bacteria(1) are trapped in the sediment allowing the clean water to collect in a storage tank. Groundwater is the largest supplier of fresh water for consumption and processing. Unfortunately, due to agriculture demands the water from underground aquifers is depleting faster than it can recharge(7). It is essential for the future of industrial agriculture that stormwater is properly handled, and aquifers are sustained. Bioretention basins are one approach to mitigating this issue(1) and can be especially beneficial in urban environments where disturbed landscapes prevent water from returning to the ground.
Another affirming quality, bioretention basins function to preserve the health of urban ecosystems increasing the overall species diversity of an area by preventing flooding, restoring urban aquifers, and reducing contamination. Often, heavy metals collect on the surface of the basin because of its function as a filter for groundwater and runoff. Despite this, bioretention basins have higher species richness, abundance, and diversity than other greenspaces(5). According to the International Journal of Landscape Science, Planning, and Design this a result of gravel, increased plant biomass, and a higher amount of leaf litter(5).
Typically, bio-basins are filled with native vegetation which creates a habitat for pollinator species, invertebrates, fungi, protists, and bacteria. High soil moisture(3) and moist leaf litter contribute to stable ground temperatures which create a habitat for earthworms(4), springtails(4), amphipods(4), mites(4), and aquatic organisms(4). The large amounts of leaf litter provide habitats for decomposer species. In turn, the release of nutrients by decomposers increases the pH5 of the soil which prevents soil acidification. Without a thick layer of leaf litter and a thriving invertebrate population, the soil is prone to acidification(5) through leaching(6), a process where rainfall strips the upper soil layer of nutrients over time. Soil acidification increases the solubility of aluminum(6) which is toxic(6) to plants, microbes, and invertebrates. In addition, soil acidity inhibits nutrient absorption6 in plants and decreases bacterial activity(6). However, a healthy and well maintained bioretention basin has the capacity to support a diverse ecosystem.
Ecosystems rich in biodiversity are more stable over time because higher levels of diversity encourage resilience to extreme weather, disease, and changing environmental conditions. Measuring the degree of biodiversity can help indicate the overall health of an ecosystem. Simpson’s Index of Diversity is calculated by sampling species richness – the number of species present in an environment(13) and species abundance – the number of individuals present in an environment within each species(13). To assess the health and diversity of the bioretention basin on American University’s campus this study will report these categorizations of species diversity for all plants, protists, bacteria, and animals.
Many techniques will be used to study biodiversity within this bioretention basin. Plants and vertebrates can be visually observed and identified with minimal intrusion into the transect. Evidence shows that the properties of an ecosystem are most often determined by its dominant plant species(14). The bioretention basin is groomed seasonally and there is reason to believe that its plant species were placed intentionally. Vertebrates and invertebrates alike are irreplaceable actors in the fixing and maintaining carbon in ecosystems(15). Invertebrates within the transect are identified by placing representative leaf and soil samples into a Berlese Funnel(13). A dichotomous key, or a self-guided tool that compares two morphologies at a time(13), is used to determine the species of an unknown individual. Dichotomous keys are also used to identify the protists and algae collected using a Hay Infusion Culture(13). Using agar to plate serial dilutions of the Hay Infusion Culture provides observable bacterial and fungal species. From there, these species can be categorized using gram staining and observations of colonies. The antibiotic tetracycline(13) is used on half the agar plates to screen for antibiotic resistance. In addition to organism observations, soil conditions such as pH, light exposure, and moisture within the transect are also measured at three representative locations because of their previously stated importance to ecosystem integrity. Lastly, the weather in the transect will be monitored and its influence on the presence of species will be accounted for.
The objective of this study is to observe and categorize the ecosystem of the bioretention basin as accurately as possible with limited intrusion. Fundamentally, bioretention basins are filters for polluted water that flow through surrounding environments. They have extensive leaf and soil cover, high moisture levels, and vegetative varieties. Based on our knowledge and current observations we hypothesize that these factors increase biodiversity within American’s bioretention basin ecosystem.
Methods and Materials
Species Diversity, Richness, Abundance
A total of 11 grams of soil/ground was collected from multiple locations in our transect. This sample included dirt, soil, mulch, and twigs. The sample was placed in a jar with 500mLs of Deerpark water and 0.1 grams of dried milk. Then the contents of the jar were shaken for 10 seconds to mix. The culture was left uncovered and undisturbed for 1 week before samples were taken to locate protozoa.
Inoculated nutrient agar Petri dishes were prepared in order to observe and quantify the prokaryotic organisms living in the Hay Infusion culture. Tetracycline agar Petri dishes were also inoculated to observe if the bacteria residing in the Hay Infusion culture is antibiotic-resistant. Since bacteria are extremely abundant, if prepared without dilution it would be impossible to characterize and count all of the colonies on the dish, thus the agar Petri dishes were prepared with serial dilutions of the hay infusion. For both the nutrient and tetracycline agar Petri dishes, they were prepared at the diluted rates of 10^-3, 10^-5, 10^-7, and 10^-9. To prepare the diluted agar plates four tubes of 5 mL sterile broth were labeled as 10^-2, 10^-4, 10^-6, and 10^-8. Then, the hay infusion culture was mixed and 50 µLs of liquid were removed and added to the tube labeled as 10^-2 and then swirled to create a 1:100 dilution. From here, 50 µLs of the 10^-2 dilution was added to the tube labeled as 10^-4 and then swirled to create a 1:10,000 dilution. This was repeated two more times to make the 10^-6 and 10^-8 dilutions. To plate the diluted broths, 100 µL of liquid was pipetted from the 10^-2 onto the 10^-3 plates and then was spread evenly. This was repeated with 10^-4 onto the 10^-5 plates, 10^-6 onto the 10^-7 plates, and 10^-8 onto the 10^-9 plates. The plates were then incubated, agar side up, at room temperature for one week.
To determine if the bacteria in the hay infusion cultures were gram-positive or gram-negative, four bacterial samples were prepared for gram staining. Using a sterile toothpick a tiny amount of the bacterial culture was scrapped from the surface of the agar plate and mixed into a drop of water on a glass slide. This was done for two nutrient agar cultures and two tetracycline agar cultures, all were labeled respectively. The slides were heat-fixed by holding them bacterial smear side up for several minutes above a flame. Once dried the bacterial smears were placed in a staining tray and covered with crystal violet for 1 minute and then immediately rinsed off with water. Next, the bacterial smears were covered with Gram’s iodine for 1 minute and then immediately rinsed off with water. Then the bacterial smears were decolorized by flooding with 95% alcohol for 10-20 seconds. They were then rinsed underwater, during which the solvent flowed colorless from the slides. The smear was then covered with safranin stain for 20-30 seconds and rinsed with water. Excess water was blotted from the side of the slide with a kimwipe and then the slide was given time to air dry. To view the stained bacteria the slide was first focused on the 10x objective lens and then viewed under the 100x oil immersion objective.
To prepare the Berlese Funnel 25mL of a 50:50 ethanol/water solution was poured into a 50mL conical tube. A screen was fitted into the bottom of a funnel and then a gallon of leaf litter was placed into the funnel. The funnel and the conical tube were secured to a ring stand so that the base of the funnel is held to the opening of the tube. Then parafilm was applied where the funnel and the tube connected to prevent the evaporation of the ethanol solution. A 40-watt lamp was placed above the funnel, 2 inches from the leaf litter, to drive the invertebrates away from the heat and into the tube below. Everything was covered with foil to keep the temperatures hot and then left alone for 1 week.
The area of land that is being monitored and cataloged for ecological life is a 20-foot by 20-foot area of land. This transect is a biorention basin with a busy road to its southeast and buildings on its three other sides. The substrate consists of soil, mulch, and leaf litter.
Image 1: Aerial diagram of transect with plant locations labeled as a-k. A, B, and C are the locations where soil conditions were measured.
The transect contains a multitude of abiotic and biotic features. Some of the abiotic features include three large rocks, two smaller rocks, a metal drain, and a metal/plastic sign. The area of all the abiotic features was combined and divided by the total area of the transect to determine the percentage of the transect covered by abiotic features. This value was calculated to be 13.4%.
% Transect covered by abiotic features: (53.65 ft2 of abiotic features / 400 ft2 transect)*100 = 13.4125% (excludes soil/mulch).
Biotic features include plants and fungi. The area of all the biotic features was combined and divided by the total area of the transect to determine the percentage of the transect covered by biotic features. This value was calculated to be 13.4%.
% Transect covered by abiotic features: (17.5 ft2 of abiotic features / 400 ft2 transect)*100 = 4.375% (excludes soil/mulch).
Three locations within the transect (A, B, and C) were chosen to monitor the moisture, pH, and light exposure of the environment.
Table 1: Soil conditions of locations A, B, and C within the transect. Measurements were all taken at a 45-degree angle facing northwest.
The soil characteristics are displayed in Table 1. The average moisture of the transect was determined to be 7.35, the average pH is 6.99, and the average light exposure is 1364. The weather conditions were recorded on these days as well.
Table 2: Weather conditions for the transect during the days of outside activity.
The weather conditions were recorded for February 7, 2019 and February 14, 2019 in Table 2. For these days there was on average 10.64 hours of sunlight, an average high temperature of 52.5 F, 45.5 F, the atmospheric pressure was 30.11inHg, the wind was 4mph, the relative humidity was 58%, precipitation was 0.05in, the visibility was 10 miles, and the UV index was 2.
Table 3: Raw data characterizing the plant species that live in the transect.
The transect contains 11 species of plants and 46 individual plants, all of them are angiosperms with monocots being more abundant. Specifically, the transect contains: 2 Magnolia virginiana, 24 Calamagrostis acutiflora, 5 patches of an unknown ground plant/weed, 1 Fallopia japonica, 2 Amelanchier intermedia, 2 Sambucus, 2 patches of Acorus calamus, 3 Swida sericea, 1 patch of an unknown grass, 2 Hydrangea quereifolia, and 2 Prunus serrulata. This data, along with descriptions of each species is displayed in Table 3.
In order to understand the biological diversity of the plants in the transect, the species richness, species abundance, and species diversity index was calculated for the plants in the transect.
Figure 1: Displaying the calculations for species richness, abundance, and diversity index of plants in the transect.
For the plants in this transect, the species richness is 11, the relative species abundance is 0.3048, and the species diversity index is 3.28. The calculations for these values is displayed in Figure 1.
Hay Infusion Observations:
- The jar has no odor.
- Residue on top of jar due to the evaporation of liquids.
- Oil-like film on the surface of the water. Twigs floating at top of jar.
- Brown sediment at bottom of the jar, some material coming up the sides of the wall.
- Thin whitish growth on twigs at bottom of jar and some reddish-tan film as well.
- Most of the twigs and plant material sunk to the bottom of the jar.
- Water has a brownish tint and is murky.
Image 2: Displaying Niche’s 1, 2, and 3 from the Hay Infusion culture.
The hay infusion culture was divided into three niches.
Niche 1- Surface of the jar. The area with oily-film and floating twigs. Area is 2mm (h) x 82mm (d). Approximately 10.5mL in volume.
Niche 2- Area between surface and bottom of the jar. No twigs, plants, etc.
Niche 3- Bottom of the jar, including sediment, plant matter, and twigs. Area is 8mm (h) x 82mm (d). Approximately 42.2 mL in volume.
We decided to analyze Niche 1 and 3 because they have the most organic matter under a microscope in hopes of identifying any living organisms.
Table 4: Raw data characterizing the protist species that were found in Niches 1 and 2 within the Hay Infusion culture.
Examination of 0.5mL of water from Niche 1 revealed 20 Euplotes, 1 Neourostylopsis, and 13 Stylonychia, which are all species of ciliate protozoans. Niche 1 also contained 70 Synura algae colonies. Since Niche 1 is approximately 10.5mL in volume we estimated that the entire niche contains 420 Euplotes specimens, 21 Neourostylopsis, 243 Stylonychia, and 1470 Synura algae colonies. These values were used to calculate the protozoan species richness, abundance, and species diversity index.
Figure 2: Displaying the calculations for species richness, abundance, and diversity index for protozoan species in Niche 1 of the Hay Infusion Culture.
For the protozoan living in Niche 1 of this transect, the species richness is 3, the relative species abundance is 0.5046, and the species diversity index is 1.98. The calculations for these values are displayed in Figure 2.
An examination of 0.5mL of water from Niche 1 revealed 80 Chlamydomonas, a species of protozoan, and 200 single-celled unidentified algae. Since Niche 3 is 42.2mL in volume we estimate that the entire niche contains 6,752 Chlamydomonas and 168,000 single-celled algae. These values were used to calculate the protozoan species richness, abundance, and species diversity index.
Figure 3: Displaying the calculations for species richness, abundance, and diversity index for protozoan species in Niche 3 of the Hay Infusion Culture.
For the protists living in Niche 3 of this transect, the species richness is 1, the relative species abundance is 1, and the species diversity index is 1.98. The calculations for these values are displayed in Figure 3.
A comparison of these two niches indicates that Niche 1 has overall higher biodiversity. This is supported because Niche 1 has more species richness than Niche 3 (3 > 1) and Niche 1 has more species diversity than Niche 3 (1.98 > 1). Although it is important to note that Niche 3 has more species abundance than Niche 1 (1 > 0.5) because it only had one species.
Table 5: Displaying the colonies of prokaryotes that live in the Hay Infusion Culture.
The hay infusion culture was used to incubate bacteria on agar plates. After one week of incubation the 10^-3 nutrient plate contained 176 prokaryotic colonies (3,520,000 colonies/mL), the 10^-5 nutrient plate contained 10 prokaryotic colonies (20,000,000 colonies/mL), the 10^-7 nutrient plate contained 0 prokaryotic colonies, the 10-9 nutrient plate contained 0 prokaryotic colonies, the 10^-3 nutrient/tetracycline plate contained 23 prokaryotic colonies (460,000 colonies/mL), the 10^-5 nutrient/tetracycline plate contained 0 prokaryotic colonies, the 10^-7 nutrient/tetracycline plate contained 0 prokaryotic, and the 10^-9 nutrient/tetracycline plate contained 1 prokaryotic colony (20,000,000,000 colonies/mL). From the colonies/mL values of the nutrient agar plates we estimated that the hay infusion culture contained a total of 5,880,000,000 prokaryotic colonies. This data, along with descriptions of each agar plate is displayed in Table 6.
Table 6: Characterization of bacterial/fungal colonies from the Hay Infusion culture.
The prokaryotic colonies from the agar plate cultures were characterized and 12 different species were identified. The majority of these colonies were opaque and punctiform/small in size. This data, along with detailed descriptions of each colony is displayed in Table 6.
Table 7: Displaying the species of bacteria that make up some of the colonies from the Hay Infusion Culture.
Bacteria from the Hay Infusion culture which were cultured on nutrient agar were gram-negative bacillus while bacteria cultured on the tetracycline agar were composed of gram-positive and gram-negative coccus as well as gram-negative bacillus. The tetracycline antibiotic was effective at killing bacteria, especially gram-negative and bacillus shapes, which is why the antibiotic plates contain fewer colonies. However, some bacteria have previously developed resistance to the antibiotic. These antibiotic-resistant bacteria are larger in size than their counterparts and many were gram-positive. This data, along with descriptions of each agar plate is found in Table 7.
Table 8: Characterization of invertebrate species.
The transect contains 1 millipede, 1 garden grub, 5 ants, 1 woodlouse, 1 spider, a different species of garden grub, and 1 dermestid beetle larvae. All of these invertebrate species are members of the Arthropoda phylum, and insects were the most abundant of the subphylum. This data, along with descriptions of each species is displayed in Table 8.
In order to understand the biological diversity of the invertebrates in the transect, the species richness, species abundance, and species diversity index was calculated for the transect.
Figure 4: Displaying the calculations for species richness, abundance, and diversity index for invertebrate species in the transect.
For the vertebrates living in this transect, the species richness is 11, the relative species abundance is 0.256198, and the species diversity index is 3.903. The calculations for these values are displayed in Figure 4.
Table 9: Characterization of vertebrate species.
The transect contains 2 Sciurus carolinensis, which are members of the Mammalia phylum. The transect also contained 2 species of Aves: 1 Cardinalis cardinalis, and 5 Turdus migratorius migratorius. This data, along with descriptions of each species are displayed in Table 9.
In order to understand the biological diversity of the vertebrates in the transect, the species richness, species abundance, and species diversity index was calculated for the transect.
Figure 5: Displaying the calculations for species richness, abundance, and diversity index for vertebrate species in the transect.
For the vertebrates living in this transect, the species richness is 3, the relative species abundance is 0.4688, and the species diversity index is 2.133. The calculations for these values are displayed in Figure 5.
To quantify the similarities and differences of the vertebrates found in this transect and the ecosystem of a different transect the Jaccard’s Coefficient of Similarity equation was used.
Jaccard’s Coefficient of Similarity
Transect X: 2 Eastern Gray Squirrels, 1 Northern Cardinal, 5 American Robins
Transect Y: 2 Eastern Gray Squirrels, 7 Koi Fish
a=2, b= 6, c= 7
The Jaccard Coefficient of Similarity value for these two transects is 0.13, meaning that these ecosystems are 13% similar.
After completing several different tests such as calculating species diversity, richness, and abundance, creating a hay infusion to determine different types of protozoa found, making a serial dilution to find different forms of bacteria, completing a gram stain to determine if the bacteria were gram-negative or positive which helped us differentiate between bacteria with gram-positive (stain violet) bacteria that have more peptidoglycan, or negative (stain pink) and have less peptidoglycan, and using a Berlese funnel to determine different invertebrates in the transect area. These tests helped us to find conclusive data on the different species inhabiting the American University Bioretention Basin. Overall, each test was successful in finding several different species such as protozoa, bacteria, plants, and invertebrates and in helping us identify these different species.
This experiment is important because it studies not only speciation within bioretention basins but also provides an example of a seemingly successful food web. Each organism in the basin holds symbiotic relationships with another, a crucial factor for the diversity and health of an ecosystem. The food chain begins with primary producers such as plants and algae. Plants and algae produce their own food photosynthetically, growing leaves and stems for themselves and other species around them. Plants also produce oxygen as a byproduct. Primary consumers such as ciliate protozoa, grub bugs (beetle larvae), and ants receive and store nutrients from consumption. Secondary consumers such as frogs obtain a part of the energy stored by the insects. Neourostylopsis, one of the ciliates identified, was substantially larger than other protozoa identified. It is possible that Neourostylopsis consumes smaller protists or is more efficient at obtaining nutrients. Tertiary consumers are birds, mice, and rats. These animals eat secondary consumers, and primary consumers to obtain energy. The final group in the food chain are higher-level consumers. Examples of these species would be predatory birds such as eagles(17). We did not encounter any higher-level consumers in the transect. In addition to all of these levels of consumers and produces are bacteria. Overall, there are several ecosystems in the transect and they all interact to produce a healthy and functioning environment.
All of the plants identified within the bioretention basin are angiosperms, and the majority are dicots. The bacteria provided by the serial dilution of the hay infusion had some intriguing qualities. For example, the colonies grown on the nutrient agar plates were significantly smaller than the colonies grown in the presence of tetracycline. The only invertebrates found were Arthropoda and the majority of those were insects. The protists were mostly ciliates and the larger protozoa were found in smaller quantities. In addition, niche 1 in the hay infusion contained the most life, possibly because of its proximity to an oxygen source.
One difficulty encountered during the investigation of biodiversity within the transect was identifying plant species. Most observations of the bioretention basin were during January and February. Many plant species lacked leaves and other defining characteristics because of the time of year. Additionally, the AU’s bioretention basin is groomed seasonally and kept tidy. Evidence of recent trimming and pruning also inhibited identification.
If time and funds permitted, conducting an analysis of the bio-basin’s ground cover and soil would be important for further interpretation of the species present. As previously mentioned, it is suspected that the bioretention basin has heavy metals present in its soil because of its function as a filter for run off. A microwave-assisted digest can report the presence and concentration of 15 heavy metals present in soil or fertilizer(18) such as iron, lead, and mercury.
Similarly, more frequent and varied pH samples within the transect would provide a more concrete analysis of the soil properties. Additionally, exploring the pH levels at varied locations on AU’s campus would provide a useful comparison of soil properties with and without drainage systems. Both testing for heavy metals and analyzing pH would provide for a better investigation into the health of the transect. Lastly, the ability to gather data for a longer period of time, possibly during two or more seasons would also greatly enhance our understanding of the transect and its overall species diversity.
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