Natural Science Seminars - Abstracts

Fall 2004

Kesi Stoneking
September 20, 2004
Effects of growing conditions on essential oil composition of sweet basil (Ocimum basilicum)
Mentors: Dr. Dean Kahl and Dr. John Brock

Abstract: Sweet basil (Ocimum basilicum L.) is a popular culinary herb that is prized for its distinct aroma and flavor, which is largely due to volatile essential oils. Previous research has shown that the environment affects the essential oil composition of basil. The objective of this study was to determine the effects of hydroponic cultivation and field cultivation on the essential oil composition of sweet basil. Basil was grown in both greenhouse hydroponic systems and field plots. After four weeks the basil was harvested and 10 samples were collected for each treatment. The essential oils for each treatment were isolated by steam distillation. The samples were analyzed by gas chromatography with a flame ionization detector (GC-FID). An unpaired t-test with a Welch correction was used to analyze the four main constituents of basil essential oil. Significant differences were found for all components analyzed: eugenol (P=<0.0001), cineole (P=0.0003), methy chavicol (P=<0.0001) and linalool (P=0.0003). Field cultivated basil contained larger amounts of eugenol, cineole, and linalool than the hydroponic basil. The main component in hydroponic basil was methyl chavicol which was present in very small amounts in the field basil. This research shows that there is a significant difference in composition of essential oil in field and hydroponic cultivated basil. From this analysis, however, it cannot be determined which cultivation method produces basil with the most desirable essential oil. Further research needs to be done on how these constituents contribute to the flavor of basil.

Samantha A. Sizemore
September 20, 2004
Determination of Total Mercury in Lake Julian Sediment
Mentor: Dr. Victoria Collins

Abstract. Coal fired power plants are the major anthropogenic source of mercury in the environment. Since mercury can cause neurological and developmental defects, monitoring mercury levels in populated areas is important. This study compared mercury concentration of sediment in Lake Julian, adjacent to Asheville Coal Power Plant operated by Progress Energy, to mercury concentration in the unpolluted Swannanoa River. Five sediment samples were collected from deep water sites in Lake Julian near the power plant. Six comparison samples were collected from the Swannanoa River at Warren Wilson College. Mercury levels of sediment were tested using EPA procedures 7471A and 7470A, based on the absorption of ultraviolet radiation by mercury vapor. Sediment Hg concentrations were compared to EPA standards. The mean mercury concentration of Lake Julian sediment was 1.93 ppm. The standard error of the mean of Lake Julian sediment was +/- 2.50 ppm. The mean mercury concentration of Swannanoa River sediment was 0.47 ppm. The standard error of the mean of Swannanoa River sediment was +/- 0.09 ppm. Mercury concentrations in Lake Julian sediment were more variable, suggesting possible pockets of contamination, but the two sites did not differ significantly in average mercury concentration (p=0.47).

Cayce Canfield
Chemical Analysis of the Pigeon River: Past and Present
September 27, 2004
Mentor: Dr. Victoria Collins

Abstract: The pollution of the Pigeon River by Champion Paper Mill (now Blue Ridge Paper Products) has been a topic of debate for almost one hundred years. Several chemical and biological studies have been conducted over the years on Pigeon River to determine the impact of the paper mill. This study is not only important due to the aesthetic value of the river, but also for the wildlife and their habitat in and around the Pigeon River. The objectives of this descriptive study were to compare the chemical aspects in the Pigeon River above versus below the Blue Ridge Paper Product’s Mill and to compare the 2004 chemical study to the 1973 study completed by UNCA. Ten different chemical aspects were sampled at five sites (two above the paper mill and three below) on three separate dates. The chemical aspects were quantified using one of the following methods: a standardized meter, the same method used in 1973, or a standardized Hach procedure. The results are shown in the chart below.

		Average concentration at site
		Up Stream from Mill		Down Stream from Mill
Chemical Aspect	Units	-7.1 Km	-2.1 Km	0.3 Km	6.7 Km	9.8 Km
Ammonia	mg / L	2.228	2.042	1.330	0.908	2.207
Conductivity	mMOHs	15.033	15.333	272.333	371.333	266.667
Dissolved Oxygen	mg / L	11.700	10.833	10.933	9.733	10.167
Lignins	mg / L	0.254	0.336	2.201	1.954	1.565
Nitrates	mg / L	1.923	2.324	1.362	1.042	2.885
Percent Saturation	%	120.086	110.951	119.493	103.724	113.666
pH	pH unit	7.500	7.333	7.633	7.600	7.833
Phosphates	mg / L	0.065	0.020	0.010	0.010	0.010
Sulfides	mg / L	2.031	2.569	1.673	2.509	2.270
Temperature	degrees C	17.233	16.900	20.067	20.333	20.967

The chemical aspects differed in the Pigeon River up stream from the paper mill compared to chemical aspects down stream of the paper mill. The 2004 data showed improvement in chemical aspects from the data from 1973.

Omar Barnaby
The Recovery of chemicals from the Pyrolysis of Type 7 Plastic Waste
October 4, 2004
Mentor: Dr. Dean C. Kahl

Abstract: This project was designed to determine whether type 7 plastic could be pyrolyzed to make useful chemicals. Currently only 21 % of plastic waste is recycled in the United States and number 7 plastics make up a small percentage of the amount of plastics recycled. Del-Monte ketchup bottles and Tropicana orange juice bottles were obtained from the Warren Wilson College waste stream. Vacuum distillation was used to pyrolyze the plastics. Vacuum distillation was used to remove the air from the system and to prevent combustion. Vacuum distillation was also used to isolate the pyrolysate. The pyrolysate was analyzed using PNMR, IR, and GC/MS. The results suggest that the pyrolysis product consisted largely of alkenes with 12 – 15 carbons. The results further suggest that the pyrolysate was a copolymer of polyethylene and polypropylene.

Ahliae Toulouse
Distribution of Exotic Invasive Species in the Bat Cave and Rumbling Bald Preserves
October 4, 2004
Mentor: Dr. Greg Ettl

Abstract: Invasive species have been present throughout North America since the early 18^th century. If left uncontrolled, these species will cause disruption in the natural balance of ecological productivity, and out-compete native vegetation. The most common in the western N.C. region are the following: Ailanthus, Microstegium, Wineberry, Multiflora Rose, Japanese Honeysuckle, and Oriental Bittersweet. This study is based on a random sampling of certain target forest communities (rich cove, montane oak/hickory, pine/oak heath) in Bat Cave and Rumbling Bald nature preserves with the objective being to determine the extent of invasions in these areas. The randomization was generated by a system of gps coordinates, and plots of 10m x 20m were established at these locations. An inventory was taken at each plot recording factors such as forest type, aspect, canopy closure, slope, and presence of exotics. A total of 54 plots sampled yielded 24 invaded locations, with at least one of the previously mentioned species present. A categorical data chi-squared test was used for statistical analysis. All 10 of the rich cove locations sampled were invaded, contributing to a p-value of 0.03, which suggests that the forest type is a significant factor in relation to the presence of invasives. However, when aspect was taken into account, the p-value obtained was 0.6, indicating that the null hypothesis of no difference cannot be rejected. From these results, it appears that rich cove forests should be the highest priority for treatment of invasive species in these preserves. Although, due to the low number of repetitions, further research on random plots needs to be done.

Laura Mills
October 25, 2004
A comparison of effectiveness among Lonicera japonica pollinators on Warren Wilson College Campus
Mentor: Dr. Amy Boyd

Abstract: Japanese honeysuckle (Lonicera japonica) is an invasive exotic woody vine native to eastern Asia that relies on cross-pollination to sexually reproduce. Introduced to North America in 1806 it has since spread to approximately 38 of the fifty United States with current control methods having little effect. The objective of this study was to determine which insects were pollinating the Japanese honeysuckle most effectively by studying resulting fruit set and seed set numbers in hopes of better understanding the natural history of the plant. Flowers were screened using mesh bags until the flowers opened and single pollinator visits could be observed. A total of 82 flowers were visited by pollinators Bombus spp., Apis spp., Vespidae, Diptera, Lepidoptera and Sphingidae. Fruit set and seed set numbers from each flower were then recorded. Chi-square analysis was performed between each pollinator regarding fruit set and a significant difference was found for Bombus spp. vs. Lepidoptera (P<0.025) and for Diptera vs. Lepidoptera (P<0.05). An ANOVA was run on seed set numbers resulting in a P-value of 0.0003 considered extremely significant. A Tukey-Kramer multiple comparison test of seed set numbers resulted in Bombus ssp. vs. control (P<0.001) and for Lepidoptera vs. control (P<0.01), both comparisons considered significant. Previous research on WWC campus has shown Bombus spp. to be the most effective pollinators focusing on pollinator deposition. My study concluded Lepidoptera to be the most effective pollinator of L japonica looking at fruit set and seed set.

Deja Lizer
November 1, 2004
Salamander Abundance within three Streams at Warren Wilson College.
Mentor: Dr. Amy Boyd

Abstract: Salamanders have been used in many scientific fields such as physiology, cell biology, evolutionary biology, and genetics. Studies have shown that there is less variation among studies on salamanders than any other amphibian group. This means that there can be more confidence in the projection of trends over time concerning population and species numbers. The purpose of this study was to complete an observational study to determine whether relative density of salamander populations differs between streams. I also wanted to determine if any environmental factors were influencing salamander density within streams. Focusing on three streams, I measured out 2m plots and measured stream width and depth, air and water temperature, water velocity, pH, recorded time of day, and took three measurements on each side of the bank to record its height at 0, 1, and 2m of the plot length. The results were analyzed using the ANOVA followed by Tukey-Kramer Multiple comparison test, unless they did not meet ANOVA’s assumptions, then the non-parametric Kruskal-Wallis test followed by Dunn’s comparison test was used. Regression analysis was also used to determine if the measured variables had any influence over salamander abundance. The results of the ANOVA/Kruskal-Wallis show that the three streams are statistically significant greater than expected by chance. However, regression analysis shows that Jensen was the only stream where the measured variables had a significant relationship over salamander abundance. The factors found to influence salamander abundance were pH, width, depth, and air temperature. Perhaps since Jensen is an overall smaller scale stream, microhabitats are more important to salamander success whereas in other streams they appear not to be.

Winborne H. Evans

November 8, 2004

The Antibacterial Activity of Honey from Apis mellifera

Mentor: Dr. Jeff Holmes

Abstract: Five different honeys were tested for their ability to inhibit the growth of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium. The honeys used were two manuka honeys, Warren Wilson College Mountain Honey, and two pasture honeys from Florida. An artificial honey, a complex sugar solution that mimicked the composition of honey, was used as a control. In a second experiment honeys that had been treated by either exposure to sunlight for two weeks or boiling for thirty minutes were tested. Honeys had different antibacterialactivities. The results indicate that those different bacterial species have different sensitivities to honey treatment suggest that the antibacterial activity of honey is complex and due to multiple factors. The osmolarity and pH of honey probably contributes only a small amount to the antibacterial activity, while hydrogen peroxide production and the floral nectar components are probably the more important factors. The second experiment suggeested exposure to light and heat degrades the antibacterial properties of honey. Warren Wilson College Mountain Honey exhibited the antibacterial activity equivalent to laboratory tested manuka honeys, except that it was much more sensitive to boiling. The results suggest that special attention needs to be paid to the conditions of storage and processing of honey intended for medicinal use. The method used appears to offer an inexpensive and practical method to quantify the antibacterial activity for Warren Wilson College’s future production of honey.

Katherine Reilly
November 8, 2004
Antioxidant Capacity of North Carolina Red Wine
Mentor: Dr. Victoria Collins

Abstract: Recent research suggests that antioxidants play a key role in health by protecting cells against oxidative damage cells caused by free radicals. Free radicals are the highly reactive byproducts of oxygen metabolism. Antioxidants are compounds that allow the body to scavenge and neutralize free radicals by donating one of their electrons to the free radical, thus ending the oxidation reaction. Recent studies indicate that consumption of red wine on a regular basis reduces the risk of coronary heart disease and atherosclerosis, and this benefit is ascribed to the antioxidant properties of the polyphenolic compounds found in red wine. The objective of my research was to evaluate the antioxidant capacity in four different bottles of red wine made from grapes grown in North Carolina and the brands were cabernet franc, cabernet sauvignon, madison lee, and merlot. This study used the method developed by Brand-Williams et al, which estimates free radical scavenging (FRS) capacity by the rate of consumption of the colored radical 2,2, Diphenyl-1-picryhydrazyl (DPPH). The kinetics of the reaction of wine with DPPH were determined as well as the amount of DPPH reduced by a given volume of red wine. Cabernet Sauvignon and Madison Lee reduced the concentration of DPPH significantly faster than merlot but total FRS capacity did not differ among the wines tested. The total antioxidant capacities of the North Carolina red wines were comparable to the capacity of French red wine. One serving of North Carolina red wines tested had vitamin C equivalent antioxidant capacities (VCEAC) between 350-400 mg.

Jennifer Carolyn Wilson
November 15 2004
Fungal endophyte ( Neotyphodium coenophialum) infection in tall fescue grass (Festuca arundinacea) on Warren-Wilson College farm
Mentor: Dr. Victoria Collins

Abstract: Tall fescue grass ( Festuca arundinacea) covers more acreage in the United States than any other introduced grass. Fescue is popular for its long growing season, drought tolerance, adaptability to poor soils and variable pH levels. The problem with fescue is the fungus that lives intercellularly within the plant. This endophyte, Neotyphodium coenophialum, produces toxic alkaloid chemicals that inhibit the health of mammals that graze endophyte infected fescue grass. In this descriptive study, 25 random samples were taken from six selected WWC grazing fields. Grass samples were stained with analine blue and examined by a compound light microscope. The percent infected samples are as follows Charlie’s 84%, Daisy Hill 76%, Night Pasture 28%, Stokes field 56%, College View 60% and the Horse Field 68%. Management decisions can be made to reduce the deleterious effects of the fungal toxins and the data from this study may help as a starting point.

William Pierzala
November 15, 2004
The Use of Diurnal Oxygen Curves to Study Pollution in the Pigeon River
Mentor: Dean Kahl

Abstract: The Pigeon River flows through western North Carolina. The Blue Ridge Paper mill is located in Canton, NC and has historically been a major polluter of the Pigeon River. Dissolved Oxygen can be used to indicate a river’s ability to degrade pollution at a microbial level (Langbein 1967). Diurnal oxygen curves show dissolved oxygen vs. time. Fluctuations in the curves are the result of photosynthesis, respiration, and diffusion in the water (Odum 1956). The objective of this study was to use diurnal oxygen curves to estimate the relative rates of photosynthesis, respiration and diffusion.

Also, to use data collected in 2004 to compare with data collected in 1990. Two sites were chosen and measurements were taken starting at midnight at 3-hour intervals for 24- hours. A paired T-test was conducted to compare dissolved oxygen concentrations and temperature above and below the mill at each time interval for both the 2004 and 1990 data. The results of the test indicated a significant difference in dissolved oxygen concentrations above and below the mill for both sets of data, with a p-value of 0.0053 in 2004, and a p-value of <0.0001 in 1990. A paired t-test was also conducted on temperature for both sets of data. The results indicate a significant difference above and below the mill with a p-value of <0.0001 for both 1990 and 2004. Upstream of mill in 2004, photosynthesis was estimated to be 3.5ppm/O₂/day where respiration was estimated to be –9.1ppm/O₂/day. This result indicated a higher respiration rate than photosynthesis meaning diffusion is the primary reason oxygen is present in the water. The analysis was deemed invalid because of difficulties in calculating a reaeration coefficient. This made determining whether dissolved oxygen was present from photosynthesis or diffusion impossible.

Ruth Secor
November 22, 2004
Tigers in captivity: a study of the effects of visitor presence
Mentor: Dr. Jeff Holmes

Abstract: Captive environments in zoological parks often cannot allow animals to carry out natural behaviors because of spatial constraints and negative visitor reactions. Zoos depend on normal behavior by animals to successfully achieve their goals of education, conservation, research, and entertainment, and thus, many enrichment programs have been implemented, altering variables to promote species-typical behaviors and to decrease stress. However, one variable that cannot be altered is the presence of human visitors. It appears to be intuitively obvious that human presence has an effect on captive animals, but whether this effect is stressful or not is unknown. Behavior observations combined with fecal hormone monitoring may be a successful measurement of stress. Tigers are infamous for their pacing behavior and present a difficult case for zoos because of their large natural range and predatory hunting behaviors. The objectives of my research were (1) to determine the frequency of occurrences of exploratory, resting, and stereotypic behaviors, and to examine the relationship between these tiger behaviors and human variables (visitor and keeper presence), and (2) to establish and validate Drs. Erich Möstl and Rupert Palme’s enzyme immunoassay (EIA) for measuring fecal glucocorticoid metabolites in tigers, and to determine whether fecal glucocorticoid levels fluctuate with zoo visitor numbers. The results of the behavioral aspect of my study showed that tigers spent the majority of their time displaying normal behavior, exploring and resting (95.4%), and less time displaying stereotypic behavior (4.6%). A nearly significant negative correlation was found between visitor presence and resting behavior (p<0.07) and a significant positive correlation between visitor presence and stereotypic behavior (p=0.001862). No other significant correlations were found between behaviors and human presence. The EIA could not be established and, thus, my second objective could not be achieved. However, the tigers rested less and displayed stereotypic behavior more when visitors were present, indicating this variable should be addressed when developing enrichment programs.

Cedarose Siemon
November 29, 2004
Salmonella Contamination and Antibiotic Resistance on Pasture Poultry and Conventional Poultry Farms
Mentor: Dr. Jeff Holmes

Abstract: In this study I set out to measure Salmonella contamination on nine conventional and nine pasture poultry farms, to test whether pasture poultry farms have higher levels of contamination, as has been suggested because antibiotics aren’t used. I collected thirty fecal droppings from each farm and cultured each sample separately, for the presence of Salmonella. I found that out of the samples I collected a farm had either no Salmonella or more than 50% of the samples were contaminated with Salmonella, suggesting that Salmonella is widespread if present in a flock. Consequently farms were scored as either contaminated or not. There were four out of nine conventional farms contaminated with Salmonella and two out of nine pasture poultry farms contaminated with Salmonella. There was no statistical difference in number of farms contaminated for each farming type. However, contrary to previous beliefs, no evidence was found showing pasture poultry farms were more likely to be contaminated with Salmonella than conventional farms. Each Salmonella isolate from each Salmonella positive sample, was tested for antibiotic resistance to twelve antibiotics. The antibiotics tested were twelve antibiotics of human health and veterinary importance. The antibiotic resistance results showed only one strain isolated from one conventional farm was resistant to one antibiotic: streptomycin. A single streptomycin resistant isolate was found on one of the two contaminated pasture poultry farms, while the other pasture poultry farm (P2) had 25 out of 28 Salmonella isolates resistant to streptomycin and tetracycline. This was unexpected because antibiotics are not used on the pasture poultry farms. One more isolate from farm P2 was resistant to three antibiotics and was intermediately resistant to ciprofloxacin. Ciprofloxacin resistance is rare in Salmonella. Overall very little antibiotic resistance was found.These results suggest that Salmonella contamination is not a serious problem on pasture poultry farms, but that the problems of contamination and antibiotic resistance are not simple and require further study.

Lindsay Renbaum
December 6, 2004
Chronic Soy Administration Effects on the Metabolism of Phytoestrogen Daidzein
Mentor: Dr. John Brock

Abstract: Isoflavones, a subclass of phytoestrogens, are biologically active constituents of leguminous plants demonstrating potential chemopreventative properties. Quantitation of isoflavones in human urine is necessary to better understand the activity of isoflavones. Daidzin, the most abundant isoflavone in soy products, is first metabolized in the large intestine by b-glycosidase into the estrogenic daidzein aglycone and then further conjugated with sulfate and glucuronic acid in the liver. Urinary concentrations of free and conjugated daidzein are used to determine the extent of metabolism of daidzein in individuals. The purposes of this study were to develop a method for daidzein quantification, to determine the effect of soy supplementation on the urinary concentrations of daidzin metabolites and to attempt to explain patterns in the urinary profiles of the individuals. Four female subjects were challenged with 34 mg daidzein supplements for 30 days and urinary levels of conjugated daidzein before supplementation, days 1-3 of supplementation and days 28-30 of supplementation were determined using HPLC–UV/Vis. Method development for the determination of daidzin metabolites in urine was successful. Almost all daidzein is conjugated in the liver before excreted into the urine. Urinary levels of conjugated daidzein appeared to increase during the first three days of soy challenge. However, considerable inter-individual variation occurred over 1 month of soy challenge. Statistically significant changes in daidzein metabolism did not occur over a 30-day dosing regimen (p=0.83). Metabolism of phytoestrogen daidzin varies considerably between individuals. Those who metabolize daidzein rapidly may experience nausea. Daidzein conjugation may decrease bioavailability of daidzein.

Amber Boles
January 24, 2005
Microbial Community Structure at High Altitudes in North Carolina as Indicated by Phospholipid Fatty Acid Composition.
Mentor: Dr. Victoria Collins

Abstact: Dead trees and air pollution atop Mount Mitchell taint the formerly picturesque view. Acidic fogs enclose Mount Mitchell 75% of the year, depositing concentrated air pollutants. However, the connection between acid precipitation and tree health remains unclear. Previous studies have focused on the effects of acid precipitation on foliage and soil-root interactions. No studies have examined the effects of acid precipitation on the soil microbial community structure, essential to nutrient cycling and tree health.

Phospholipid Fatty Acid Analysis (PLFA) examines microbial lipid membranes to provide information useful in assessing shifts in community structure and ecosystem functioning. Using PLFA, this study compared patterns in microbial community structure in Red Spruce (Picea rubens) soils where trees were classified as dead or alive. Soil samples were classified as “dead” or “alive” based on crown: height ratio, an indication of tree health and vigor. A healthy tree has a higher crown: height ratio (>50%).

The fungal to bacterial fatty acid ratio was compared using a t-test. Soils near dead trees had higher (P = 0.0036*) fungal: bacterial fatty acid ratios than soils near alive trees, indicating different microbial community structures. No differences in soil color, as measured by value and chroma, and soil pH were found between living and dead stands (P = 0.451, P = 0.197, P = 0.357 respectively). While the soil microbial community structure differs between living and dead stands, this study cannot determine whether changes in soil microbial composition precede or follow tree death.