UCNJ Fall 2023 Research Journal

UCNJ U N I O N C O L L E G E O F UNION COUNTY, NJ Undergraduate Research Journal Volume 6 | No. 1 | Fall 2023

UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6 | No. 1 | Fall 2023 Editorial Team: Editor-in-Chief Mohamed Mohamed, Ph.D. Director of Student Research and Science Laboratories Associate Editor Melissa Sande, Ph.D. Associate VP of Academic Affairs & Dean of Humanities Shuchi Agrawal, Ph.D. Assistant Dean of STEM Olubisi Ashiru, Ph.D. Academic Specialist, Biology William Dunscombe Dean of STEM Amjed Hedhli, COE. Academic Specialist, Computer Science Sunjin Jo, Ph.D. Academic Specialist, Chemistry Nabil Kabakibi Academic Specialist, Engineering Susana Sequeira, AIA Academic Specialist, Architecture Faraz Siddique, Ed.D. Associate Dean of STEM Shahrzad Taghdissi Academic Specialist, Chemistry

1 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023

2 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1| Fall 2023 MESSAGE FROM THE EDITOR Zora Neale Hurston, the renowned American anthropologist, once described the research as “Formalized curiosity. It is poking and prying with a purpose.” This quote describes what our undergraduate researchers do and why. This journal represents UCNJ's commitment to the promotion of undergraduate research. Since 2019, this journal has served as a showcase for the diverse research of our students. UCNJ firmly believes that engaging in research helps students acquire essential research techniques while the journal provides a forum for students to publish their work. I am pleased to present this issue of the UCNJ Undergraduate Research Journal. This issue is a testament to the dedication and hard work of our student researchers, faculty mentors, lab staff, and the College administration. The articles in this volume of the journal span a wide spectrum of STEM topics including ethical hacking, testing the antibacterial action of bioactive compounds extracted from the trees on the UCNJ campus, exploration of anti-microbial and anti-fungal properties of essential oils, the use of DNA barcoding for plant identification, the synthesis of Acetylsalicylic acid compound, and Napoleon’s brother attempts to grow trees in Bordentown, New Jersey. The multidiscipline articles in this issue represent the breadth and depth of research in community colleges. The President of UCNJ, Dr. Margaret M. McMenamin, and the Provost, Dr. Maris Lown, have made student research a priority. It should also be noted that undergraduate research would not be possible without devoted faculty members, academic specialists, and lab staff members who have worked alongside the students, guiding, and mentoring them throughout their research journey. I am confident that the articles in this issue will contribute to the scholarly community. Sincerely, Mohamed Mohamed, Ph.D. Director of Student Research and Science Laboratories UCNJ Union College of Union County, NJ

3 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1| Fall 2023 Using Wireshark: Ethical Hacking and Network Defense Kayla Lugo, Hunza Khan, Felix Pinzon Gaona, Angel Tomas Mentor: Academic Specialist Amjed Hedhli STEM Division, UCNJ Union College of Union County, NJ Abstract - A considerable portion of the US population remains unaware of the serious threats posed by keylogging, which can have severe consequences for both large corporations and individual families. To protect sensitive data from these attacks, we must educate ourselves on how hackers use keyloggers, how to detect them on our devices, and how to safeguard against them. In this research, a web-based application that mimics a login terminal and utilizes a C# keylogger to simulate an enterprise-level malware attack was developed. When the keylogger captures the user's keystrokes, we will leverage Wireshark, a powerful opensource network traffic monitor, to collect and analyze the recorded data. It is essential to note that all research and testing undertaken as part of this study were conducted within ethical guidelines. Introduction As the internet continues to rapidly evolve and progress, cybersecurity is struggling to keep pace with the growing number of skilled hackers and significant challenges to our country's cyber intelligence. In the United States, there were 1862 data breaches in 2021, surpassing the previous record of 1506 breaches set in 2017 (Ball, 2022). Keyloggers were the sixth most common type of enterprise malware among these data breaches (Vale, 2023). Keylogging, also known as keystroke logging, captures and records every keystroke made on a keyboard. Despite being in use for decades, many internet users are unaware of keyloggers, their ease of use, and the speed with which they can compromise an individual's entire identity. To study this phenomenon, we created a web-based application that appears to be a login terminal but will have a C# keylogger running in the background. As the "user" inputs information, we will monitor their keystrokes using the keylogger and collect and monitor data using Wireshark. This research provides the experience of being an ethical hacker for a day, understanding how this hack is executed, and learning how to protect ourselves and our systems from similar hacks. This research aimed to raise awareness of the dangers of keylogging, as it is not a widely recognized hacking technique among the public. Materials and Methodology This study involved the development of a webbased application that masquerades as a login terminal, with a C# keylogger integrated into it. Wireshark was utilized to gather and supervise data. The online keylogger, commonly referred to as a "web-based keylogger" or "web-based keystroke logger," is a keylogger that functions via a website or web application. Figure 1: Space Network testing environment

4 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Furthermore, every keystroke entered on the website is captured and recorded. The logged data is then sent via email. Results and discussion 1. Significance One of the main risks of keyloggers is that they can be used to capture sensitive information, such as login credentials, passwords, and credit card numbers. If a keylogger is installed on a computer, it can record everything typed on the keyboard, including sensitive information. This information can then be accessed by the person who installed the keylogger. Another risk of keyloggers is that they can be used to monitor a person's activities without their knowledge. This can be particularly concerning if the keylogger is installed on a personal computer, as it may record private conversations, emails, or other sensitive information. Keyloggers are the tools of choice for most hackers due to the lack of expertise required to create them. Creating a keylogger allows us to glance through a hacker’s lens and helps improve our cybersecurity skills to defend against these kinds of attacks. Most individuals are unaware of how keyloggers work and how to spot one on a device since they are installed without the user’s consent or knowledge. In August of this year, a former public utility employee of a Northern Ohio company pleaded guilty to installing keylogger devices on the company’s computers (Chin, 2023). FBI agents interviewed the former employee (John Pelton) to gain insight (Chin, 2023). This instance is among the various keylogger security breaches this year. Keyloggers are a growing phenomenon; to be prepared, we must understand how they work. 2. User Interaction To execute our research successfully, the user will interact with the interface by entering a username and password (Figure 1). The hacker will not gain any access to the device/their personal information without the user inputting anything. This is critical to the attack. 3. System and Functional Requirements For WireShark to be utilized effectively, it is necessary to have the basic knowledge of how packets are sent throughout a network as well as the difference between transmission control protocol (TCP) and user datagram protocol (UDP) packets. Our team also learned how to filter and extract packages on Wireshark. For this web application to be successful, it must allow the user to access the created account web page by clicking on the created account hyperlink. Next, this webpage must allow the user to log in and access the webpage by using the correct username and password. Figures 2 and 3: Website login screen and Website sign-up screen.

5 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 4. External Interface Requirements For optimal performance, this web application requires a device that has at least 4GB of RAM with an i5 or higher, It is recommended to have a designation in order to stay up-to-date with the constantly changing internet landscape. As cybersecurity concerns mount due to the everincreasing number of skilled hackers, our nation’s cyber intelligence is facing significant challenges. Vale (2023) reported that in 2021, there were 1862 data breaches in the United States, which is higher than the 1506 breaches reported in 2017. Among the types of malware used, keyloggers were ranked sixth and were frequently used in enterprise attacks. Figures 4 & 5: Flowchart of the keystroke logging process and our project progress. Keylogging, also known as keystroke logging, is a method used to capture and record every keystroke made on a keyboard. It is essential for the smooth operation of web applications. In terms of the Graphic Processing Unit (GPU), it can be used in both recent and older models, allowing users to display web applications efficiently. In this study, a switch, which is a network device that connects multiple devices, will be used. The switch receives packets of data from connected devices and forwards them to their intended destination. When a packet is received by the switch, it examines the packet header to determine the MAC address of the destination. The switch then uses this information to forward the packet to the correct device on the network (Vale, 2023). The keylogger must be skillfully crafted to function seamlessly across different processing units such as CPU, RAM, and GPU. It will be developed using the programming language C# and designed as a web-based tool. Once the web-based keylogger is installed, it can record all user input, including sensitive data like login credentials and credit card information. The captured information is usually stored on the web server and can be accessed by the website owner or an attacker who has gained access to the server. Web-based keyloggers can monitor user activity, acquire confidential information, and conduct malicious activities like phishing and identity theft. 5. Nonfunctional Requirement For the web application, the nonfunctional requirements are performance and compatibility, Figure 6: Set up of a network.

6 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Figure 7: Setup of a network. reliability, availability, and security. For compatibility as specified in external requirements, certain conditions must be met for this application to run smoothly. Availability refers to the accessibility of an application to users who are testing it. In Figures 6 and 7, we are showcasing the successful setup of a network environment. This involved skillfully configuring a switch, ethernet cables, and a router. To achieve this, we connected the switch to the router using ethernet cables and then linked our PCs to the switch. This resulted in automatic IP address assignment through Dynamic Host Configuration Protocol (DHCP).). Notably, the victim's laptop had the IP address 10.2.221.142, while the hacker's PC was assigned 10.3.221.139. To verify the network connection, we utilized the Internet Control Message Protocol (ICMP) protocol to successfully ping both devices within the same network. Figure 8: Logged keystroke data to the hackers Figure 9: Logged keystroke data to the hackers. Upon execution, the key logger effectively sends the logged keystroke data to the hacker's email as a .txt file. As a result, the hacker was then able to gain unauthorized access to our webpage using the credentials "Admin" and "UCC123". Maintaining a website goes hand in hand with security as it allows developers to make updates based on prior security breaches or update how securely the website functions. As was seen in this research, updating this web page becomes crucial to securing all usernames and passwords (HTTP vs. HTTPS). For the keylogger, the hacker will make nonfunctional requirements based on the kind of network their victim resides in and on the vulnerability of the target. Changes can be made if penetrating/executing the file becomes a problem. This will increase efficiency. The team dedicated a significant amount of time to troubleshooting the project. During troubleshooting, we conducted extensive tests and made several adjustments to the JavaScript and C# code to ensure that it integrated smoothly with the web application. Our primary focus was to make sure that the user's login credentials were stored securely and reliably in a text document. To efficiently search for specific packets, we utilized Wireshark's filtering tool, leveraging keywords and the TCP/IP/SMTP protocol to identify the packets required for analysis.

7 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Figure 10: Data breach after Cyberattacks. The image illustrated in Figure 10 presents compelling evidence that a cyber-attack had transpired, leading to a data breach. To unravel the intricacies of this attack, our Cyber Incident Response team member conducted a meticulous investigation. Their findings revealed that the hacker had sent a deceitful email to one of the individuals with website access, subsequently executing a keylogger. Once the victim clicked the email, the hacker gained access to the admin's login credentials after infiltrating the victim's computer. To determine how the sensitive data was transmitted to the hacker, we employed the use of Wireshark, a powerful tool for logging and analyzing network traffic. The captured data revealed a suspicious Simple Mail Transfer Protocol (SMTP) packet originating from an IP address flagged as 10.3.221.39, which provided a critical lead in the investigation. Conclusion After conducting a thorough analysis of the website's front end, which includes HTML, CSS, and JavaScript, and an extensive evaluation of the C# keylogger implemented on the back end, the research provided valuable insights into the operations of keyloggers and effective defense mechanisms against them. Future research will focus on advancing skills to capture, decipher, and filter data packets using Wireshark to enhance troubleshooting and prevention efforts. Future Work Given the opportunity to conduct additional ethical tests of our keylogger on multiple computers, our team will expand our knowledge base. By decrypting the transmitted data and reverse-engineering the keylogger, insights will be gained into the identity and origin of the sender, representing a prime example of cyber security defense. Acknowledgment This research was made possible by the National Science Foundation, IRAP grant 1832425. Its contents are solely the award recipient’s responsibility and do not necessarily represent the official views of the National Science Foundation. Contact information. Amjed Hedhli: [email protected] Angel Tomas: [email protected] Felix Gaona: [email protected] Hunza Khan: [email protected] Kayla Lugo: [email protected] References Ball, I.L. Former Public Utility Employee Pleads Guilty to Installing Keylogger Devices on Work Computers. (2022, August 10). https://www.justice.gov/usao-ndoh/pr/former-public-utilityemployee-pleads-guilty-installing-keylogger-devices-work Chin, Kyle. Biggest Data Breaches in US History (Updated 2023): Up guard. RSS. (N.D.). Retrieved March 20, 2023, from https:/www.upguard.com/blog/biggest-data-breaches-us Stouffer, W. by C. (n.d.). Keyloggers 101: A definition + keystroke logging detection methods. Norton. Retrieved March 20, 2023, from https://us.norton.com/blog/malware/what-is-akeyloger#:~:text=In%20some%20form%20or%20another,a%20suite%20of%20Trojan%20viruses Vale, Bryan. Learning center - what is the network layer - tutorial - cloudflare (n.d.). Retrieved March 30, 2023, from https://community.cloudflare.com/t/learning-center-what-isthe-network-layer/1788

8 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Preliminary Screening of Magnolia stellata Stems and Malus sylvestris Bark for Antibacterial Activity Vivianna L. Hodges, Gabriela M. Duran, and Jada A. Jordan Mentor: Academic Specialist Dr. Olubisi Ashiru STEM Division, UCNJ Union College of Union County, NJ Abstract – The preliminary screening of Magnolia stellata stems, and Malus sylvestris bark for antibacterial activity involved the collection of each plant’s parts from their trees located on the UCNJ Union College of Union County, NJ Cranford Campus. This was subsequently followed by the maceration of the dried tree plant parts in two solvents, petroleum ether (C6H14) and ethyl acetate (C4H8O2). The petroleum ether and ethyl acetate were used respectively to extract nonpolar and polar constituents of the dried tree plant parts. The tree plant extracts were assessed for their antibacterial properties against two bacteria species Staphylococcus epidermidis and Escherichia coli. Zones of inhibition observed on the bacterial lawns of S. epidermidis, and E. coli were indicative of potential antibacterial activity in the tree plant part extracts. Introduction The use of plants for medicinal purposes has long existed and continues to gain popularity, with researchers providing scientific data to support the benefits of using plant parts to treat illness (Salmeron-Manzano et al., 2020). Plants could be used as alternative remedies to address the issue of side effects from drug toxicity and the development of resistance to the drugs. The encouragement and promotion by the World Health Organization (WHO) for the development and use of medicinal plants has also resulted in more screenings of plants for their antimicrobial properties (Mickymaray et al., 2016). Magnolia stellata commonly called “star magnolia” is an ornamental Japanese native plant that grows in Europe and America (Callaway, 1994). The bark of Magnolia officinalis (a member of the same genus as Magnolida. stellata) has been used to treat gastrointestinal disorders. The plant was reported to have antimicrobial activity against Staphylococcus aureus (a member of the same genus as Staphylococcus epidermidis) (Hu et al., 2011). Malus sylvestris commonly called “crab apple” is a deciduous European native tree (Cornille et al., 2014). M. sylvestris has been reported to have antimicrobial properties (Hunter & Hull, 1993). The Cranford campus of UCNJ Union College of Union County, NJ has many beautiful trees, some of which have been cataloged (Ombrello, 1997). The presence of these trees contributes to improving the air quality on the campus. Furthermore, these trees provide shade for both staff members and students to sit under on hot summer afternoons. Are there more benefits that these tree plants located on the Cranford campus can provide, besides the aesthetic layout of the grounds and improved air quality? Do some of these tree plants have antibacterial activity? This study was analyzed to preliminary screen extracts from the bark and stem of two tree plants found on Cranford campus of UCNJ Union College of Union County, NJ for antibacterial activity against two bacteria species (Staphylococcus epidermidis and Escherichia coli). Two different solvents, petroleum ether (C6H14), a nonpolar solvent and ethyl acetate (C4H8O2), a polar solvent were used to target non-polar and polar constituents of the tree stems and bark. Materials and Methods A. Collection of Plant Parts Stems of M. stellata and bark pieces of M. sylvestris tree plants (Figs 1A & 1B) were collected from the trees on the Cranford campus of UCNJ Union College of Union County, NJ. The tree plant

9 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 parts were rinsed with tap water and kept in separate trays to air dry (Figs. 2A & 2B). Figures 1A & 1B. Magnolia stellata (common name - Star Magnolia) and Malus sylvestris (common name Crab apple) growing on the campus of UCNJ Union College of Union County, New Jesey. Figures 2A & 2B. Stems of Magnolia stellata and Barks of Malus sylvestris. B. Preparation of Extracts The air-dried tree parts were weighed. 50g of the air-dried stems of the M. stellata tree plant were placed in an Erlenmeyer flask and 200mL of petroleum ether (C6H14) was added. This setup was repeated, but ethyl acetate (C4H8O2) was used as the solvent, in place of petroleum ether. Fifty grams of the air-dried bark of M. sylvestris were placed in an Erlenmeyer flask and 200mL of petroleum ether (C6H14) was added. This setup was repeated, but ethyl acetate (C4H8O2) was used as the Figure 3. Macerated tree parts solvent, in place of petroleum ether. The stems and bark were macerated and left for eight days in each flask (Fig. 3). Thereafter, the obtained extracts were filtered and the filtrates from each flask were stored separately in dark bottles for later use. C. Inoculum & Disc preparation Separate bacterial lawns of S. epidermidis and E. coli were made on Tryptic soy agar plates. Each plant extract filtrate was separately aliquoted into labeled sterile 5mL glass beakers. Sterile blank discs (6mm) were individually dipped into each tree plant extract filtrate. The impregnated discs were placed onto the separate bacteria lawns of S. epidermidis and E. coli. Sterile blank discs were also separately dipped into either petroleum ether or ethyl acetate and included as control references. All plates were incubated for 24 hours at 37oC. Results and discussion No zone of inhibition was observed around the control reference disc impregnated with petroleum ether, while a zone of inhibition was observed around the control reference disc impregnated with ethyl acetate (Figs. 4A, 4B, 5A & 5B). Zones of inhibition were observed around all the discs impregnated with petroleum ether-based tree extract filtrates (ranging from 6mm – 10mm) and ethyl acetate-based tree extract filtrates (ranging from 7.7mm – 9mm) on both bacteria lawns of S. epidermidis and E. coli (Figs. 4A, 4B, 5A & 5B). Figures 4A & 4B. Bacteria lawns of S. epidermis and E. coli respectively, show zones of inhibition around discs impregnated with petroleum etherA B A B A B

10 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 based tree plant extract filtrate, ethyl acetatebased tree plant extract filtrate, and only ethyl acetate. Figure 5A. Antibacterial activities of petroleum ether-based tree plant extracts of M. stellata stems and M. sylvestris bark against S. epidermis and E. coli. Figure 5B. Antibacterial activities of ethyl acetatebased tree plant extracts of M. stellata stems and M. sylvestris barks against S. epidermis and E. coli. Preliminary results showed that both M. stellata stems and M. sylvestris bark extracts express potential antibacterial activities against both E. coli and S. epidermidis. Petroleum ether was used to target the extraction of the non-polar bioactive compounds from M. stellata stems and M. sylvestris bark extracts because of its polarity of 0.117 (Abubakar & Haque, 2020). Ethyl acetate was used to target the extraction of polar bioactive compounds from M. stellata stems and M. sylvestris bark extracts because of its polarity of 0.228. The rotary evaporator used for this experiment did not have a chiller. Therefore, it could not evaporate the two solvents (petroleum ether and ethyl acetate) from the filtrates obtained after the maceration of the plant’s stem and bark. To address this issue, we used discs dipped either only in petroleum ether or ethyl acetate as control references to benchmark our results. This was done to ensure that any observed zone of inhibition around the discs was not due to the solvent’s activity against the bacteria. However, in the case of the solvent - ethyl acetate, zones of inhibition were observed around the control reference discs (Fig. 5B). However, slightly wider zones of inhibition were observed around the discs impregnated with ethyl acetate-based tree plant extract filtrates on both bacteria lawns of S. epidermidis and E. coli. Due to what was observed using ethyl acetate, there may be a need to consider using another solvent with high polarity to extract polar bioactive compounds from the tree plant parts extracts. Conclusion Our results agree with the results confirmed by previous reports about M. sylvestris having antimicrobial properties (Hunter & Hull, 1993). The observation about M. stellata adds new knowledge to previously published work on M. officinalis (Hu et al., 2011). Future Work Future work could include investigating the antibacterial activity in the leaves and flowers of both M. stellata and M. sylvestris. Phytochemical screening will be done on tree-part extracts that show increased antibacterial activity. This will be done to detect the presence of secondary metabolites (e.g. alkaloids, terpenoids, coumarin, tannins, saponins, fatty acids, etc.). Future research will include applying the same techniques to obtain extracts from other tree plant species found on the Cranford campus of UCNJ Union College of Union County, NJ. Acknowledgment This research was made possible by the National Science Foundation, IRAP grant 1832425. Its contents are solely the award recipient’s responsibility and do not necessarily represent the official views of the National Science Foundation.

11 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 We also thank Dr. Thomas Ombrello, Dr. Mushtaq Khan, Ms. Karen Ryan, Mrs. Beata Mourad, Ms. Tamiko Carman, and UCNJ Union College of Union County, NJ STEM Division, for their continuous support. Contact information Dr. Olubisi Ashiru: [email protected] Vivianna L. Hodges: [email protected] Gabriela M. Duran: [email protected] Jada A. Jordan: [email protected] References Abubakar AR, Haque Preparation of medicinal plants: basic extraction fractionation procedures for experimental purposes. (2020). J Pharm Bioallied Sci. 12(1):1-10. https://doi.org/10.4103/jpbs.JPBS_175_19. Callaway D. J. (1994). The world of Magnolias. Timber Press: Oregon, USA; 260pp. Cornille A., Giraud T., Smulders M. J. M., RoldánRuiz I., & Gladieux P. (2014). The domestication and evolutionary ecology of apples. Trends Genet. 30:57-65 Hu Y., Qiao J., Zhang X., & Ge C. (2011). Antimicrobial effect of Magnolia officinalis extract against Staphylococcus aureus. Journal of Science and Food Agric. 91(6):1050-6. https://doi:10.1002/jsfa.4280 Hunter MD, Hull LA. Variation in concentrations of phloridzin and phloretin in apple foliage. (1993). Phytochemistry 34:1251-4 Mickymaray, S., Al Aboody, M. S., Rath, P. K., Annamalai, P., & Nooruddin, T. (2016). Screening and antibacterial efficacy of selected Indian medicinal plants. Asian Pacific Journal of Tropical Biomedicine, 6(3):185–191. https://dx.doi.org/10.1016/j.apjtb.2015.12.005 Ombrello TM. The Trees of Union County College. 1997 2nd Edition Salmerón-Manzano, E., Garrido-Cardenas, J. A., & Manzano-Agugliaro, F.(2020). Worldwide Research Trends on Medicinal Plants. International Journal of Environmental Research and Public Health (IJERPH), 17(10):3376. https://doi.org/10.3390/ijerph17103376

12 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Anti-Microbial and Anti-Fungal Properties of Essential Oils Wilmer Aldair Guevara, Sophia Zayas, Juliana Herrera Mentor: Academic Specialist Shahrzad Taghdissi STEM Division, UCNJ Union College of Union County, NJ Abstract—Due to the emergence of new diseases, cleanliness through disinfection or germicides is important in the prevention of sickness. Natural resources like essential oils have great potential to be more accessible for their anti-microbial and anti-fungal properties. Additionally, this research provides information about the properties of essential oils and their potential anti-microbial and anti-fungal effects on common surfaces. Introduction The prevalence of drug-resistant microbes and fungi has increased over the years. This has led to increased concern about the ability of medical and social environments to maintain cleanliness in the face of rapidly evolving microorganisms. Natural resources such as essential oils have a wide range of uses in promoting better hygiene practices to combat these issues. Essential oils may serve as an alternative to antibiotics and antifungal agents. During the winter, it can be particularly challenging to maintain cleanliness on surfaces in high-traffic areas. In an experiment conducted at UCNJ Union College of Union County, NJ, microbial growth was swabbed in several locations such as cafeterias, chemistry labs, college libraries, and bathrooms. This research aimed to address the effectiveness of essential oils in eliminating microbes that inhabit common surfaces. Essential oils from Melaleuca alternifolia (Tea Tree), Jasminum gandiflorum L. (Jasmine plant), Cymbopogon citratus (Lemongrass), Eucalyptus globulus (Eucalyptus), Origanum vulgare (Oregano), Salvia rosmarinus (Rosemary) were used in this research. These oils have medicinal properties and are safe for skin contact. They were tested to eliminate surface microbes. Microorganisms and fungi are commonly found on almost all surfaces. Fungi thrive in environments with optimal humidity and temperature for their spores to grow. Microbes and fungi can multiply rapidly in the right conditions. Many essential oils have hydrophobic properties that distort the phospholipid bilayer of cell membranes, leading to cell death and inhibition of propagation. To test their effectiveness, different concentrations of essential oils were evaluated. These oils are soluble in alcohol, ether, and fixed oils but not in water. Terpenes are one of the significant compounds found in most oils. Terpenes are primarily found in plants and offer specialized chemical interactions and protection in abiotic and biotic environments. The most effective antimicrobial terpene is found in thyme essential oil. Figure 1: Bioactive compounds in essential oils

13 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Bioactive compounds found in essential oils are crucial in combating both fungal and microbial resistance. Fungi are notoriously difficult to eliminate due to their high resistance to antifungal agents (Gao et al., 2020). This is because their cellular structure makes them resilient. The diversity of microbes and their constant evolution can result in the creation of resistant offspring. To provide a safer and more accessible option to traditional cleaning products, essential oils can be utilized. This research was to identify the most effective concentrations of specific essential oils that could eliminate or impede the growth of microorganisms. Materials and Methods A. Preparation of Agar Plates For the experiment, powdered nutrient agar that was diluted with one liter of deionized water was used. The solution was heated in an autoclave for 30 minutes to an hour and then let cool to room temperature for about 30 minutes. After that, the solution was poured into 30 plates and allowed to cool. The agar provides an optimal environment for the growth of the microbes. Figure 2: Creation of agar plates after cooling B. Microbe Growth Microorganisms were collected from various areas at UCNJ Union College of Union County, NJ, including the chemistry lab, bathroom, cafeteria, and campus library. A plastic streaker was used to swab agar plates, which were then incubated for to allow for the growth of microbes and fungi. Figure 3: Culture grown from a different location. After isolating the microbes growing on the plates, we observed the effects of oils on cultures with varying concentrations. We separated the oils by creating divisions on new agar plates, where the cultures were grown. Figure 4: Testing the oils. To assess the impact of diverse concentrations of essential oils, we selected the three most potent oils - Tea tree, Rosemary, and Lemongrass - and diluted them with water to observe their ability to hinder microbial growth. Water was chosen due to its wide availability, despite its hydrophobic properties. In addition, we conducted experiments using a blend of these oils in their pure form on separate agar plates. To achieve this, we utilized microbial/fungal growth that had not been inhibited on agar plates that were minimally affected by the previously tested oils and combined the three most

14 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 essential oils to compare the effects of concentration dilution. C. Gas Chromatography A gas chromatograph was used to obtain a more detailed chemical analysis of the essential oils. A volume of 0.1 milliliters of different oils was injected into the engine, further breaking down the compounds to create a graph of peaks. This graph helps to identify similarities in the chemical composition of the oils, using retention time and characteristics. It's essential to remember that results obtained through GC may vary based on the essential oils' manufacturing and purity level. Figure 5: GC High Peak Time – 10.14 Peak High Point 927.30 mV for the tree of Tea oil. Figure 6: GC- High Peak Time- Time 3.16 Peak High Point 899.00 mV for Rosemary Oil When comparing the peaks, similarities can be observed between Lemongrass and Tea Tree oils. These similarities were found in similar locations and at the same time. On the other hand, Rosemary oil showed a significant difference in composition within its time and location. These peaks indicate the groups that are most present in the compounds. Since the oils were placed under the same conditions for the same duration, the results reveal the specific groups that make up the oil. Results and discussion Several essential oils were tested for their ability to inhibit the growth of microorganisms. The oils that showed antimicrobial and antifungal properties were Melaleuca alternifolia (Tea Tree), Cymbopogon citratus (Lemongrass), Salvia rosmarinus (Rosemary), and Origanum vulgare (Oregano). These oils produced a clear zone of inhibition on the agar plates, indicating their effectiveness in preventing the growth of microbes. On the other hand, Jasmine and Eucalyptus oils did not show any inhibition, suggesting that they have little to no effect in preventing the growth of microorganisms. Further tests were conducted using the Lemongrass and Tea Tree oils on a sample collected from the chemistry lab. The results showed that these oils were effective in preventing the growth of microorganisms, indicating that they have possible germicidal properties. It is worth noting that some oils, such as Jasmine and Eucalyptus, may not be effective in preventing the growth of microorganisms, but they are still widely used for their potential therapeutic benefits. Figure 7: The clear circle in the center shows inhibition of growth with Tee Tree oil. The final experiment was conducted to determine the effectiveness of a combination of three oils. When mixed at equal ratios of 3 drops each, Tea Tree, Lemongrass, and Rosemary oil did not

15 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 produce any significant results. Surprisingly, the concentrated mixture did not inhibit growth, even though all the oils had previously demonstrated individual germicidal effects (Tholl, 2015 & Rashed et. al., 2021). It's possible that the mixture was too concentrated to kill any microbes. These oils work by being toxic to the microbial cell wall at high concentrations, but there might be optimal concentrations that work best. Figure 8: Agar plate exposed to diluted oils. Our experiment using gas chromatography provided insights into the antimicrobial and antifungal properties of oils. The major antibacterial agent found in Tea Tree oil is terpinene-4-ol, which is also the most prominent peak in its graph. Other common peaks include 1,8-cineole, alpha-terpinene, and gamma-terpinene. High concentrations of terpinene-4-ol indicate high-quality oil, whereas excessive presence of compounds such as 1,8-cineol or alpha-terpinene may suggest contamination. According to Jalali- et al., 2023 study, Rosemary oil, with 1,8-cineole as its major component, is known for its purity and quality. Other peaks suggest compounds such as camphor, borneol, and alpha-pinene, which similarly indicate contamination. Camphor is the primary contributor to the antiseptic properties of this oil. Lastly, Lemongrass oil contained the most components. The oil's largest peak in its GC graph is a mixture of citral, limonene, geraniol, and various other terpenes. Smaller peaks include other compounds such as esters, alcohols, and aldehydes. Citral has the strongest anti-microbial activity against fungi and bacteria indicated by the diameter of the inhibition zone (Chouhan et al.,2017). Tea Tree oil demonstrated the most effective antimicrobial and anti-fungal properties after analyzing the GC graphs of all oils (Gao et al. 2020). The experiment encountered challenges in the results, that would need to be addressed if the experiment is repeated. It was difficult to determine the effectiveness of the oils because the bacteria on the agar plates couldn't be identified. Additionally, some microorganisms on the agar plates showed resistance to the inhibition, while others showed sensitivity. Given the vast and unknown range of microbes, it was difficult to determine which oil would work best against which microorganism. Conclusion Based on the available data and the results, Tree Tea, Lemongrass, and Rosemary oils have demonstrated anti-microbial and anti-fungal properties. However, as they have limited water solubility, their concentration dilution must be carefully controlled. The optimal concentration range of these oils is critical to their effectiveness and these results are consistent with the findings of Dhifi et. al., (2016) and D'agostino et. al., (2019). Essential oils have the potential to be used as natural disinfectants for various surfaces and even for medicinal purposes. Furthermore, researchers may be encouraged to explore the use of these oils in antimicrobial and antifungal resistance research to determine which oils are effective in inhibiting the growth of specific microorganisms as explained in the article published by Sciarrone et al., (2010 & 2022). Acknowledgment This research was made possible by the National Science Foundation, IRAP grant 1832425. Its contents are solely the award recipient’s responsibility and do not necessarily represent the official views of the National Science Foundation. We thank Ms. Karen Ryan and UCNJ Union College of Union County, NJ STEM Division.

16 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Contact Information Shahrzad Taghdissi: [email protected] Sophia Zayas: [email protected] Wilmer Guevara: [email protected] Juliana Herrera: [email protected] References Chouhan, S., Sharma, K., & Guleria, S. (2017). Antimicrobial Activity of Some Essential Oils—Present Status and Future Perspectives. Medicines, 4(3). https://doi.org/10.3390/medicines4030058 D'agostino, M., Tesse, N., Frippiat, J. P., Machouart, M., & Debourgogne, A. (2019). Essential Oils and Their Natural Active Compounds Presenting Antifungal Properties. Molecules 24 (20), 3713. https://doi.org/10.3390/molecules24203713 Dhifi, W., Bellili, S., Jazi, S., Bahloul, N., & Mnif, W. (2016). Essential Oils’ Chemical Characterization and Investigation of Some Biological Activities: A Critical Review. Medicines, 3(4). https://doi.org/10.3390/medicines3040025 Gao, S., Liu, G., Li, J., Chen, J., Li, L., Li, Z., Zhang, X., Zhang, S., Thorne, R. F., & Zhang, S. (2020, November 20). Antimicrobial activity of lemongrass essential oil (Cymbopogon flexuosus) and its active component citral against dual-species biofilms of Staphylococcus aureus and candida species. Frontiers. Retrieved April 5, 2023. https://www.frontiersin.org/articles/10.3389/fcimb.2020 .603858/full Jalali-Heravi, M., Sadat Moazeni, R., Sereshti, H., & Analysis of Iranian rosemary essential oil: Application of gas chromatography–mass spectrometry combined with Chemometrics. Journal of Chromatography. https://www.sciencedirect.com/science/article/pii/S00219673 11002755#fig0005 Tholl D. (2015). Biosynthesis and biological functions of terpenoids in plants. Advances in biochemical engineering/biotechnology, 148, 63– 106. https://doi.org/10.1007/10_2014_295 Rashed, Abd. A., Rathi, N. G., Husna Ahmad Nasir, N. A., & Abd Rahman, A. Z. (2021). Antifungal Properties of Essential Oils and Their Compounds for Application in Skin Fungal Infections: Conventional and Nonconventional Approaches. Molecules, 26(4). https://doi.org/10.3390/molecules26041093 Sciarrone, D., Geissler, M., & Mondello, L. (2022, April 15). Quantitative analysis of the composition of Tea Tree Oil. Chromatography Online. Retrieved April 5, 2023, from https://www.chromatographyonline.com/view/qua ntitative-analysis-composition-tea-tree-oil Sciarrone, D., Ragonese, C., Carnovale, C., Piperno, A., Dugo, P., Dugo, G., & Mondello, L. (2010). Evaluation of tea tree oil quality and ascaridole: a deep study using chiral and multi heartcuts multidimensional gas chromatography system coupled to mass spectrometry detection. Journal of chromatography. A, 1217(41), 6422.

17 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 DNA Barcoding Method for Plant Identification and 16S rRNA Analysis of Rhizosphere Bacteria Liliana Hopkins, Kayla Yim, Ana Rumora, Melissa Baykus, Luisa Martinez Mentor: Dr. Luis Jimenez Biology and Horticulture Department, Division of Mathematics, Science, and Technology Bergen Community College, Paramus, New Jersey Abstract-This article reviews the results of the barcoding of two trees (T1 and T3) and the metagenomic analysis of their rhizosphere microbial communities using next-generation sequencing of 16S rRNA genes. With rapidly advancing sequencing technologies, species identification calls for improved techniques. The first objective of this study was to develop an accurate and replicable DNA barcoding method for land plant taxonomic classification. We evaluated the ability of three genetic barcodes: the first internal transcribed spacer (ITS1) region of the nuclear ribosomal cistron, the second subunit of the internal transcribed spacer (ITS2), and chloroplast trnL (UAA) intron. The barcodes had a 50% success rate, identifying half of the samples as the same species. Metagenomic analysis of the identified trees’ rhizosphere microbiomes revealed their relative bacteria compositions. All communities showed similar demographics at the phylum, class, and genus levels. The most abundant phylum in Tree 1 (T1) was Actinobacteria and Proteobacteria for Tree 3 (T3). Alphaproteobacterial was the most abundant class in both samples. At the genus level, most species were unidentified. The most abundant genus in both samples was Rhodoplanes. These results call for replicated studies with larger sample sizes. Introduction A comprehensive understanding of the ecosystem requires integrated and dimensional experimental research. Land plant systems must be analyzed within the context of their environment. A bottom-up approach might include a study of both genetic composition and microbial community. Modern gene sequencing technologies and databases promote DNA barcoding as an efficient taxonomic classification method. The benefits of genotypic-based taxonomy include improved specificity and accuracy (Gostel and Kress, 2022). There are several potential genes to use for land plant taxonomic classification. The internal transcribed spacer (ITS1) region of the 18S–5.8S– 26S nuclear ribosomal cistron is commonly used as a fungal and plant species barcode. It exhibits the potential to be used as a universal eukaryotic barcode (Jimenez et al., 2019). Its subregion, ITS2, may be used as a plant-specific barcode (Wang et al., 2015). Studies classify this gene as a supplementary confirmation sequence (Timpano et al., 2020). A chloroplast trnL (UAA) intron can be employed with the recommended ribosomal genes to maximize accuracy and reduce potential bias. The primers have a high resolution in specific contexts, including common plant organisms (Taberlet et al., 2006). Ribosomal and chloroplast sequences, when used simultaneously, potentially create a streamlined and reliable PCR procedure for land plant identification. The biological processes of land plants must be studied about their supporting microbial communities in the rhizosphere (Lei et al., 2019). Land plants depend on their symbiotic relationships with the bacteria and fungi in their respective microbiomes. Depending on the tree species, different species of bacteria enable various essential metabolic reactions such as nitrogen fixation, phytohormone production, defense against plant pathogens, and the liberation of nutrients (phosphorus and sulfur) to sustain plant growth (Jimenez et al., 2019). Metagenomic analytical tools using 16S rRNA sequencing characterize bacterial diversity at the individual, species, and community levels (Jimenez

18 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 et al., 2020; Lei et al., 2019). An impediment that genetic analysis of bacterial communities’ faces is a lack of information in genetic databases (Liu et al., 2022). Below the class level, metagenomic statistical analysis is often uninformative. With the currently available information, studies can determine the most abundant phylum, class, and other general taxonomic levels in rhizospheres (Lagos et al., 2015). When studied in conjunction with an accurately identified and related plant, this information reveals potential functional relationships between plants and bacteria. This study focuses on land plant barcoding and soil metagenomics to build a genetic map of tree and bacterial communities, to make inferences about the functional relationships that connect them. Materials and Methods A. Site description The experiment was conducted at the laboratory of Dr. Luis Jimenez, Department of Biology and Horticulture, Bergen Community College (BCC) (40.9509° N, 74.0882° W). The average elevation of the study area was 16 m above sea level. The climate is a temperate continental humid climate. B. Tree and soil sampling Two trees around the BCC campus were studied, based on assumed phenotypic differences and location. Tree samples were defined as T1 and T3. The coordinates of the two tree samples are listed in Table 1, and pictures of each tree are shown in Figure 1. Table 1. Coordinates of T1 and T3. Tree Sample Coordinates Tree 1 (T1) (40.9501630, -74.0874088) Tree 3 (T3) (40.9512594, -74.0888753) Tree 1 (T1) Tree 3 (T3) Figure 1. Trees 1 and 3. Leaf samples were collected in a sterile container on May 24, 2023. The samples were relatively free of contaminants, such as bird fecal matter or insect larvae. Soil samples were collected through June 2023, in sterile containers, from areas adjacent to tree roots. If the roots were buried in mulch or other biodiversity-altering landscaping materials, samples were collected from at least 6 inches below surface level. C. Tree and soil DNA extraction For tree DNA extraction, “Quick-DNA” protocols from Zymo Research (Zymo Research, Irvine, CA) were followed, with minor modifications as per Jimenez et al. (2020). The leaves were cut into small pieces and then ground with a paste with mortar and pestle. 100-200 microliters of sterile water were added to accelerate the process. Leaf genomic material was extracted from 0.05-0.2 grams of the ground leaf paste. The first step in the standardized Zymo Research procedure was altered; the leaf sample was vortexed for 10 minutes to ensure cell lysis occurred. Soil genomic material was extracted as previously described (Jimenez et al., 2020). D. Primer selection and optimization All PCR reactions were performed in a biological safety cabinet to minimize the possibility of contamination. We targeted one chloroplast and two ribosomal genes to optimize accuracy, specificity, and reliability in our land-plant taxonomic classification method. The internal transcribed spacer (ITS) region of the 18S–5.8S 26S nuclear

19 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 ribosomal cistron is a potential universal barcode for eukaryotic organisms (Jimenez et al., 2019). To amplify the ITS sequence, ITS1, and ITS4 primers target an estimated 640 base pair (bp) fragment (Ferrer et al. 2001). We used the reaction constituents and conditions previously described (Jimenez et al., 2019). Its subregion, ITS2, was used as a supplementary, plant-specific barcode (Timpano et al., 2020). The ITS2F and ITS3R primers were used for sequence amplification. The ITS2 PCR reaction conditions used are described by Chen et al. (2010). This study used the chloroplast trnL (UAA) intron gene as a supplementary, plant-specific barcode. We followed the reaction conditions as previously detailed (Taberlet et al., 2007), using the c and d primers to amplify a 456 bp fragment. To analyze the quality of the extracted DNA, PCR amplification of the bacterial 16S rRNA gene was performed. The primers used were 27f and 1492r. Reaction mixtures contained 3 μl of DNA template, 1 μl of the 27f primer, 1 μl of the 1492r primer, 20 μl of sterile water, and 1 ReadyTo-Go (RTG) PCR bead (GE Healthcare, Buckinghamshire, UK). The reaction conditions were as follows: 1 cycle at 94°C for 5 min, followed by 30 cycles with a denaturation step at 94°C for 30 seconds, an annealing step at 50°C for 30 seconds, and an extension step at 72°C for 1 min, followed by 1 cycle at 72°C for 10 mins. After the PCR reaction was completed, a 1.5 kilobase (kb) DNA fragment indicated a positive reaction. We also used the 341f and 805r primers, which amplified a 465 bp DNA fragment of the 16S rRNA gene v3-v4 region. Standard PCR reaction protocols were followed, as described by Klindworth et al. (2012). PCR reaction success was visually confirmed using the FlashGel system (Lonza Inc., Rockland, ME) with FlashGel DNA cassettes containing 1.2% agarose. A FlashGel DNA marker of 100-4,000 kb (Lonza Inc., Rockland, ME) was used to determine the presence of amplified DNA gene fragments. E. Metagenomic analysis DNA sequencing reactions of the ITS1, ITS2, and UAA genes were performed by Azenta US, Inc. (South Plainfield, NJ). Homology searches were performed using the GenBank server of the National Center for Biotechnology Information (NCBI). http://blast.ncbi.nlm.nih.gov/Blast.cgi and the BLAST (blastn) algorithm (Altschul et al., 1997). Next-generation sequencing (“AmpliconEZ” service) was performed by Azenta/GENEWIZ (Azenta US, Inc., South Plainfield, NJ, USA). 16S rRNA analysis was performed as previously described (Jimenez et al., 2020). Results and discussion A. Tree identification using barcode genes. This study developed a plant-specific DNAbarcoding method for species identification, as opposed to a phenotypic-based taxonomy approach. To maximize accuracy, we targeted three different genes: the internal transcribed spacer (ITS1) region of the 18S–5.8S–26S nuclear ribosomal cistron, the second subunit of the nuclear internal transcribed spacer (ITS2), and chloroplast trnL (UAA) intron. Standard DNA extraction and PCR procedures were modified to optimize the DNA concentrations. Table 2 displays the identification of T1 and T3 using the 3 target genes. We observed a 50% accuracy rate. The DNA barcodes did not agree on T1 classification. However, ITS2 and UAA showed similar identification at the genus level. Both genes showed Quercus to be the genus that T1 belongs to but at the species level identification was not the same. Unfortunately, the ITS1 gene did not show the same results at the genus or species level. The identification by ITS1 barcoding was Fagus sylvatica. However, the three genes used for the DNA barcoding of T3 agreed at the genus and species level. T1 was identified as Acer Rubrum.

20 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Table 2. Genetic identification of trees based upon BLAST analysis of target genes. ITS1 ITS2 UAA Tree 1 (T1) Fagus sylvatica Quercus planipocula Quercus rubra Tree 3 (T3) Acer rubrum Acer rubrum Acer rubrum Eukaryotic organisms are complex; genetic inheritance patterns can be misleading. While ITS2 is a subunit of ITS1, they display different phylogenies. They are different genes and, therefore, subjected to different evolutionary pressures. We cannot rely on one genetic sequence to be used as a barcode to identify eukaryotic organisms such as land plants. ITS1 is preferred for its universality at the sacrifice of specificity (Jimenez et al., 2019). We paired the ITS primer with its subregion and the chloroplast (UAA) intron to create a reliable DNAbarcoding method. Both primers are potentially reliable plant-specific barcodes. When used simultaneously, they create a streamlined and reliable procedure for land plant identification. Human error and other confounding variables must be accounted for when considering the significance of this study. DNA extractions rely on a successful procedure, which we modified to accommodate for the complexity of eukaryotic organisms. The modifications made to standard DNA extraction protocols should be reevaluated and studied independently. Leaf samples are subject to contamination by the genetic material of other organisms. Further studies are needed to confirm the reliability of primer selection and DNA extraction method development for DNA barcoding as a tree species classification method. B. Rhizosphere bacterial communities Metagenomic analysis of the rhizosphere bacterial communities reported similar relative abundances of bacteria on all taxonomic levels. Figures 2 and 3 show the relative abundance corresponding to Trees 1 and 3, respectively. Figure 2. Top 10 bacterial taxonomic levels for T1. Figure 3. Top 10 bacterial taxonomic levels for T3.

21 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 The most abundant phylum in Tree 1 (T1) was the Actinobacteria (Figure 2). The most abundant phylum in Tree 3 (T3) was the Proteobacteria as shown in Figure 3. Alpha proteobacteria were the most abundant class in both samples. At the genus level, most species were unidentified. Upon identification, Rhodoplanes was found to be the most abundant genus in both samples. Further studies with larger sample sizes are needed to support the results. Building a map of the bacteria communities within the rhizosphere reveals potential relationships between microorganisms and adjacent land plants. Rhodoplanes are phototropic bacteria present in all microbial communities. They have been previously isolated from the rhizosphere of different plants (Singh et al., 2022). We hypothesize that they are essential to tree health; studies predict that Rhodoplanes are involved in carbon fixation (Khan et al., 2020). The direct chemical exchanges between tree species and their respective bacteria must be studied independently with an appropriate experimental design. These results also have macroscopic implications; previous studies suggest that long-term land use reduces the diversity of soil microbial communities (Sengupta et al., 2020). The use of fungicides, mulch, and other biodiversity-reducing landscaping tools on Bergen Community College’s campus may potentially contribute to the uniform soil demographic between tree species. Conclusion Land plants and rhizosphere microbial communities exist in symbiosis (Jacoby et al., 2017a); their biological processes cannot be understood without considering their relationship to each other. This study used an integrated experimental design to perform a metagenomic analysis of local trees and their respective soil microbiomes. Our first objective was to design a reliable method to identify tree species using DNA barcoding. We evaluated the ability of three genetic sequences for plant-specific classification: the first internal transcribed spacer (ITS1) region of the nuclear ribosomal cistron, the second subunit of the internal transcribed spacer (ITS2), and chloroplast trnL (UAA) intron. The barcodes had a 50% success rate, identifying half of the sample as the same species. Metagenomic analysis of the rhizosphere bacterial communities reported similar relative abundances on all taxonomic levels. This information, analyzed in the context of each other, reveals potential functional relationships between plants and bacteria. Future Work Future studies with a continued integrated approach replicated experimental procedures, and larger sample sizes may clarify the causal relationships that connect plant and microbial communities. We will also expand the microbiome analysis to fungi communities associated to plant rhizosphere. Acknowledgment The project was funded by a grant from the Department of Education of the United States of America to Hispanic Serving Institutions (HSI) for Science, Technology, Engineering, and Mathematics (STEM) education. Funding was also received through a United States National Science Foundation (NSF) Grant #0903168. Contact information. Dr. Luis Jimenez: [email protected] Melissa Baykus: [email protected] Liliana Hopkins [email protected] Kayla Yim: [email protected] Ana Rumora: [email protected] Luisa Martinez: [email protected] References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acid Research 25, 3389—3402.

22 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., Zhu, Y., Ma, X., Gao, T. Pang, X., Luo, K., Li, Y., Li, X., Jia, X., Lin, Y., & Leon, C. (2010). Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species. PloS ONE 5(1):e8613 http//doi:10.1371/journal.pone.0008613. Ferrer, C., Colom, F., Frasés, S., Mulet, E., Abad, J. L., & Alió, J. L. (2001). Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections. Journal of Clinical Microbiology, 39(8), 2873–2879. https://doi.org/10.1128/jcm.39.8.2873-2879.2001. Gostel, M. R., & Kress, W. J. (2022). The expanding role of DNA barcodes: Indispensable tools for ecology, evolution, and conservation. Diversity, 14(3), 213. https://doi.org/10.3390/d14030213. Jacoby, R., Peukert, M., Succurro, A., Koprivova, A., & Kopriva, S. (2017a). The role of soil microorganisms in plant mineral nutrition—current knowledge and future directions. Frontiers in Plant Science, 8. https://doi.org/10.3389/fpls.2017.01617. Jimenez, L., Gardner, M., Bochis, J., Jashari, T., Ellman, V., Vasquez, J., Zapata, S., Ramos, V., Choe, T., & Tazehabadi, M. (2019). Direct PCR detection, cloning, and characterization of fungi communities in soils and compost. BIOS, 90(2), 87. https://doi.org/10.1893/0005-3155-90.2.87. Jimenez, L., Kulko, M., Kim, R., Jashari, T., & Choe, T. (2020). 16S rRNA analysis of electrogenic bacterial communities in microbial fuel cells developed from temperate soils. BIOS, 91(1), 9. https://doi.org/10.1893/bios-d-18-00006. Khan, M. A., & Khan, S. T. (2020). Microbial communities and their predictive functional profiles in arid soil of Saudi Arabia. Microbial Communities and Their Predictive Functional Profiles in Arid Soil of Saudi Arabia. https://doi.org/10.5194/soil-2020-31. Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, C., Horn, M., & Glockner, F.O. (2012). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Research doi:10.1093/Nar/gks808. Lagos, L., Maruyama, F., Nannipieri, P., Mora, M. L., Ogram, A., & Jorquera, M. A. (2015). Current overview on the study of bacteria in the Rhizosphere by modern molecular techniques: A mini review. Journal of Soil Science and Plant Nutrition, (ahead), 0–0. https://doi.org/10.4067/s0718-95162015005000042. Lei, S., Xu, X., Cheng, Z., Xiong, J., Ma, R., Zhang, L., Yang, X., Zhu, Y., Zhang, B., & Tian, B. (2019). Analysis of the community composition and bacterial diversity of the rhizosphere microbiome across different plant taxa. MicrobiologyOpen. 2019; 8:e762. https://doi.org/10.1002/mbo3.762. Liu, S., Moon, C. D., Zheng, N., Huws, S., Zhao, S., & Wang, J. (2022). Opportunities and challenges of using metagenomic data to bring uncultured microbes into cultivation. Microbiome, 10(1). https://doi.org/10.1186/s40168-022-01272-5. Sengupta, A., Hariharan, J., Grewal, P., & Dick, W. (2020). Bacterial community dissimilarity in soils is driven by long‐term land‐use practices. Agrosystems, Geosciences & Environment, 3(1). https://doi.org/10.1002/agg2.20031. Singh, N., Singh, V., Nand Rai, S., Vamanu, E., & Singh, M.P. (2022). Metagenomic analysis of garden soil-derived microbial consortia and unveiling their metabolic potential in mitigating toxic hexavalent chromium. Life 12, 2094. https://doi.org/10.3390/ life12122094. Taberlet, P., Coissac, E., Pompanon, F., Gielly, L., Miquel, C., Valentini, A., Vermat, T., Corthier, G., Brochmann, C., & Willerslev, E. (2007). Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding. Nucleic Acids Res., 35(3):e14. doi: 10.1093/nar/gkl938. Epub 2006 Dec 14. PMID: 17169982; PMCID: PMC1807943. Timpano E.K., Scheible M.K.R., & Meiklejohn, K.A. (2020). Optimization of the second internal transcribed spacer (ITS2) for characterizing land plants from soil. PLoS One. 2020 Apr 16;15(4):e0231436. doi: 10.1371/journal.pone.0231436. PMID: 32298321; PMCID: PMC7162488. Wang, X.C., Liu, C., Huang, L., Bengtsson-Palme, J., Chen, H., Zhang, J.H., Cai, D., & Li, J.Q. (2015). ITS1: a DNA barcode better than ITS2 in eukaryotes? Molecular Ecology Resources. 15, 573–586. doi: 10.1111/1755-0998.12325.

23 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Synthesis of Acetylsalicylic Acid from Methyl Salicylate Nathaniel Baptista, Lucas Chudoba, Hayley Crowell, Abigail Dizon, Carine Medellus, Maria Silva, Krystal Villalobos Mentors: Dr. Vikul B Rajpara, Prof. Shahrzad Heidary, Dr. Mushtaq Khan STEM Division, UCNJ Union College of Union County, NJ ABSTRACT - Aspirin is one of the top-selling overthe-counter medications in modern times. Since the discovery of this drug, it has been utilized as both a pain reliever, as well as a treatment for cardiovascular diseases and other maladies. Aspirin is synthesized by using salicylic acid as a starting material commercially. The synthesis of salicylic acid from wintergreen oil is considered a classic method for curating natural medications. A classic Fischer Esterification Reaction under acid-catalyzed processes will be conducted to synthesize acetylsalicylic acid, otherwise known as Aspirin. The structure of the compound will be determined using FTIR spectroscopy and compared to standard literature values. Introduction Aspirin, also known as acetylsalicylic acid (2-Acetoxybenzoic Acid), has been one of the leading over-the-counter pain remedies since 1859 (Jack, 1997). It is synthesized by the chemical reaction of the key ingredient’s salicylic acid and acetic anhydride through acetylation. Salicylic acid contains a benzene ring and two functional groups-hydroxyl and carboxyl (PubChem., 2023). Acting as an anti-inflammatory, Aspirin inhibits cyclooxygenase Prostaglandin-Endoperoxide Synthase), an enzyme that catalyzes precursors that control processes like inflammation and blood flow and suppresses the creation of prostaglandin H2 and thromboxane (Pablo, 2023). Prostaglandins function as local hormones produced in the body that aid in the transmission of pain signals, regulating hypothalamic thermostat, and inflammation. Furthermore, thromboxanes, a group of lipids, are stimulants for platelet aggregation (Pablo, 2023). Acetylsalicylic acid achieves the suppression of this enzyme through the acetyl group, as it bonds covalently to an antibody Ser529 of the active site of the COX-1 enzyme (Nih.gov, 2023). It then interacts with the amino acid Arg120. As a result, Aspirin blocks the access to the hydrophobic channel to Tyr385 of arachidonic acid at the catalytic site. In turn, Aspirin stops the generation of prostaglandin H2 (Nih.gov, 2023). If taken daily, Aspirin operates as a medium to decrease the risk of cardiovascular complications, due to its anticoagulant properties (Nih.gov, 2023). In blood, the main function of certain platelets is to bond to each other and seal wounds. Sometimes they create a thrombus—a blood clot that forms on the wall of a blood vessel or in the heart when platelets stick together—leading to a stroke or heart attack. The NSAID diminishes the ability of blood to clump up into clots or in other words, blood thinning (Pablo, 2023). This property helps prevent the platelets from adhering to the walls of an artery and forming a thrombus (Pablo, 2023). Aspirin is an inexpensive and effective medication that is known to prevent diseases such as cardiovascular, Alzheimer's dementia, etc. Although there are numerous benefits to the commercial success of Aspirin, its widespread use of it is limited by its gastrointestinal side effects (Nih.gov, 2023). Research has shown that up to 50% of consumers who are prescribed Aspirin for secondary prevention, such as blood thinning, report gastrointestinal side effects after two weeks (Nih.gov, 2023). Along with this, almost one-third of the population have endoscopically viable lesions within an hour of ingestion. These gastrointestinal effects are inauspicious, as they range from dyspepsia with endoscopically normal gastric mucosa to complications of ulcers that include bleeding and perforation (Nih.gov, 2023). Methyl salicylate, both synthetic and natural, has been used for many years as a flavoring agent because of its pleasant, penetrating odor and minty flavor (PubChem., 2023). Wintergreen tea can be made from either the leaves of the wintergreen plant or the twigs and the inner bark of a

24 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 sweet and yellow birch tree; the distinctive wintergreen flavor is found in everything from root beer to mouthwash (PubChem., 2023). Medicinally, wintergreen oil has some pain-reducing properties of the other salicylates, including Aspirin. It can be absorbed through the skin, providing an astringent, but soothing sensation, and is frequently used in preparations for muscular aches and arthritis (PubChem., 2023). It is possible to hydrolyze methyl salicylate acid to produce salicylic acid. Methyl salicylate, an ester, forms water, methanol, and salicylic acid salt when reacted with a base, specifically sodium hydroxide (NaOH) (Chemistry 211 Experiment, Website 2023). Salicylic acid salt is neutralized by aqueous hydrochloric acid (HCl) to form salicylic acid. Once salicylic acid is purified, it is refluxed with acetic anhydride to form acetylsalicylic acid, or Aspirin (Chemistry 211 Experiment, Website 2023). While research has been done previously where methyl salicylate has been turned into salicylic acid, and subsequently, salicylic acid has been turned into acetylsalicylic acid, this research intends to combine the two processes. The research aims to synthesize Aspirin using salicylic acid produced through hydrolysis of natural methyl salicylate from wintergreen oil. Materials and Methods General Methods: Reagents and solvents were provided by the Chemistry Discipline of UCNJ Union College of Union County, New Jersey. FTIR spectroscopic data were collected with a PerkinElmer Spectrum 100 FT-IR and PerkinElmer Spectrum. Melting point readings for these compounds were taken using Stuart SMP10/120V/60 Digital Melting Point Apparatus. Method A (Base Catalyzed Ester Hydrolysis): 10.0 g of sodium hydroxide (NaOH) was dissolved in 50.0 mL of distilled water. This sodium hydroxide solution was added to a reflux apparatus with 4.3 mL of methyl salicylate (C8H8O3) and then heated for 20 minutes. After, the reaction mixture cooled between 50.0°C to 60.0°C. The mixture was then chilled in an ice bath. Method B (Acid-Catalyzed Hydrolysis of Salicylic Acid Salt): 6.0 M of hydrochloric acid (HCl) was cooled to about 0°C and slowly added to the mixture from method A to neutralize it, leaving the carboxylic acid (R-COOH) undisrupted. Approximately 7.0-10.0 mL of hydrochloric acid was added to acidify the solution to form crystals. Vacuum filtration apparatus was utilized to isolate the crude salicylic acid (C7H6O3). This mixture was then allowed to fully dry for 3 days. A melting point and FT-IR scan were performed and recorded to verify the purity of the sample. Method C (Recrystallization): The crude Aspirin was recrystallized using hot and cold distilled water as a solvent. Vacuum filtration was performed, and the pure salicylic acid crystals were left to dry for a week. After recrystallization, mass and melting points were determined and the IR spectrum was obtained. Method D (Esterification): 2.5 g of pure salicylic acid (C7H6O3), 5.0 mL of acetic anhydride (C4H6O3), and 5 drops of sulfuric acid (9M H2SO4) were mixed in a flask. The solution was heated to 50.0°C to 55.0°C, then cooled to room temperature. Around 25 mL of distilled water was added, and the mixture was placed into the ice bath to form crystals. Using vacuum filtration, crude Aspirin (C9H8O4) was isolated and allowed to dry completely. Mass and melting points were recorded. Method E (Recrystallization of crude Aspirin): 2.5 g of crude Aspirin, (2-Acetoxybenzoic Acid) was added to ethanol (about 8 mL) until it dissolved. The mixture was heated, and 20.0 mL of warm distilled water was added. Once the mixture reached 50.0°C to 55.0°C, it was cooled using an ice bath. The recrystallization and vacuum filtration from methods C and D were repeated. Once fully dried, the final product, Aspirin, was tested for purity utilizing both FTIR analysis and melting point, which were then compared to literature values. Reaction mechanisms: Hydrolysis and esterification were the two main reactions used in the synthesis of Aspirin. These two reactions are the exact opposite of each other. Through alkaline hydrolysis of methyl salicylate, followed by acidification, Salicylic acid, and methanol were formed. In contrast, through the

25 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 esterification of salicylic acid using acetic anhydride and sulfuric acid as catalysts, acetylsalicylic acid and acetic acid are formed. Base-catalyzed ester hydrolysis, also known as nucleophilic acyl substitution and acid-catalyzed hydrolysis of salt reactions was used to form solid salicylic acid from methyl salicylate. Sodium hydroxide was used in the base-catalyzed hydrolysis with the hydroxide group acting as the nucleophile and the carbonyl carbon acting as the electrophile due to the partial positive charge created from the polar covalent double bond between the carbonyl carbon and oxygen. A nucleophile tends to donate electrons while an electrophile tends to accept electrons. In this step, a tetrahedral intermediate was formed. By refluxing, the methoxide group was forced to leave. Due to the excess sodium hydroxide used, the salicylic acid reacted with the sodium ion to form a salt, as well as the methoxide to form sodium methoxide. Next, an acid-base reaction occurs with hydrochloric acid to form the desired product (salicylic acid) with methanol and sodium chloride as byproducts. The ester group in methyl salicylate becomes a carboxylic acid group. Vacuum filtration was utilized to isolate the crude salicylic acid. After recrystallization, pure salicylic acid was obtained. Through the esterification reaction, salicylic acid was reacted with acetic anhydride using sulfuric acid as a catalyst to yield acetylsalicylic acid (Aspirin) with acetic acid and water as the byproducts. The protonation of acetic anhydride occurs to make the carbonyl carbon more electrophilic for the following steps in the reaction mechanism. The hydroxyl group in salicylic acid causes a nucleophilic attack on the electrophilic carbonyl carbon that was formed from the protonation of acetic anhydride forcing water to leave. This water will then act as a base in the acid-base reaction to make the oxygen neutral (without the positive charge). The hydronium ion acts as an acid in the following step. In the last acid-base reaction, water will act as a base. Acetic acid is a good leaving group in this step, which will form the crude acetylsalicylic acid (Aspirin). Lastly, vacuum filtration was performed to isolate crude acetylsalicylic acid (Aspirin). Pure Aspirin was obtained through the recrystallization of the crude product. Figure 1 – Main Reaction Figure 2 – Base-Catalyzed Ester Hydrolysis Figure 3 – Esterification Reaction Figure 4 – Protonation of Acetic Anhydride

26 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Results and discussion Fourier-transform infrared spectroscopy, also known as FTIR spectroscopy, is the method we used to identify the starting (methyl salicylate), middle (salicylic acid), and final products (Aspirin). This method was applied to identify the composition of the materials and provide verification of the compounds. Each material has its own, unique IR spectrum. The FTIR is an effective tool to use specifically for this experiment to identify and qualify the compounds (Ye and Spencer, 2017). FTIR spectra have two main components: the Wavenumber (cm 1 ) on the X-axis and the Percent Transmittance (%T) on the Y-axis (FTIR Apodization, 2015). Each band on the spectrum represents the vibrational frequency of the functional groups. As for the transmittance, IR radiation is passed through the sample by the IR spectrometer. Part of the radiation is absorbed by the specific functional group of the compound and the rest is transmitted (EBSCO Information Services website, 2023). Each functional group has a specific wavenumber. The FTIR spectra measure wavenumber the specific functional groups that absorb the photons of infrared radiation. Each band represents the specific vibrational frequency at which the bonds vibrate within the functional group of the molecule (Ashenhurst website, 2023). To analyze the scans, we utilized the tabled values of IR wavenumbers provided by the University of California, Los Angeles (Ye et.al. 2019). It is imperative to emphasize these two specific regions for the IR spectra. The region from 4,000 cm-1 to 1500 cm-1 is known as the functional group region, where the wavenumbers of specific functional groups are found, such as hydroxyl and amino groups which are 3700 cm-1 - 3100 cm-1 . The region defined by 1500 cm-1 to 450 cm-1 is defined as the fingerprint region. Each functional group within a molecule has a unique bonding energy, much like a human fingerprint (Illustrated Glossary of Organic Chemistry website, 2023). Each functional group is represented on this spectrum (Figure 6(A)). The region that is 1500 - 450 cm-1 is known as the fingerprint region. The wider parts of the graph represent “stretches,” which are the increase or decrease of bond length within the functional group (Lepodise website, 2023). Represented above (Figure 6 (B)) are the numbered readings of the FTIR scan of methyl salicylate. The intense peaks with low % Transmittance are caused by the double bonds in the C=O bond as well as the aromatic carbons. Salicylic acid was formed after the hydrolysis of esters. Each functional group of salicylic acid is present within this spectrum (Figure 7 (A)). The region that is 1500 - 450 cm-1 is known as the fingerprint region. Represented in (Figure 7(B)) are the numbered readings of the FTIR scan of salicylic acid. The intense peaks with low % Transmittance are caused by the double bonds in the C=O bond as well as the aromatic carbons. Moreover, within the IR spectrum, there is a wide stretch between 3500 - 2250 cm, -1 due to the alcohols, the first being on the carboxylic acid and the second being on the phenol. Represented in (Figure 8) is our final product, Aspirin. Again, there is a wide stretch due to the alcohol group attached to the phenol. In addition, many oxygens broaden the -OH stretch more than it already was. The region that is 1500 - 450 cm-1 is known as the fingerprint region. Figure 6 (A) - FTIR Scan of Methyl Salicylate Figure 5 – Esterification Reaction

27 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Represented above are the numbered readings of the FTIR scan of acetylsalicylic acid. The intense peaks with low % Transmittance are caused by the double bonds in the C=O bond as well as the aromatic carbons. The wide stretch is caused by the alcohol group on the carboxylic acid. The oxygen also broadens the -OH stretch. The melting point apparatus was an additional instrument in this experiment. Melting point is a physical property of compounds. It is a key factor in the determination of the purity of substances, due to the uniqueness of the property. Purity cannot solely be verified through this apparatus, as multiple compounds have similar melting points. It is useful when used with other instruments. In Table 1, the melting point of each compound isolated was taken. The melting points, along with the FTIR spectrums, were used to verify the identity of the compound. synthesized after each step. Pure compounds have well-defined melting points with a range of 1℃, while crude compounds showcase a melting point with a 2℃ range, as there can be a presence of impurities that alter the melting point. Both acid and base-catalyzed hydrolysis reactions were used during the synthesis. The experiment differs from traditional Aspirin synthesis, as it begins with the naturally occurring material, the oil of wintergreen, and requires two steps rather than one. The product isolated after each step was purified and confirmed by FTIR spectroscopy and melting point analysis. With the use of both qualitative and quantitative methods, it was verified that pure Aspirin was successfully synthesized using the two-step method. Conclusion A two-step synthesis of Aspirin was performed by converting the starting material, methyl salicylate, into salicylic acid followed by esterification into pure Aspirin. Acknowledgment This research was made possible by the National Science Foundation, IRAP grant 1832425. Its contents are solely the award recipient’s responsibility and do not necessarily represent the official views of the National Science Foundation. We also thank Ms. Kathryn M. Anaczkowsski for guiding us in utilizing the FTIR. A special thank you to Professor Compound Observed Melting Point Literature Melting Point Crude salicylic acid 160-161℃ 158.6℃ Pure salicylic acid 161-162℃ 158.6℃ Crude Aspirin 137-139℃ 135.0℃ Pure Aspirin 134-135℃ 135.0℃ Figure 6 (B) - Tabulated Values for Methyl Salicylate FT-IR Scan LINK Figure 7 (B) - Tabulated Values for Salicylic Acid FT-IR Scan LINK Table 1 - Observed Melting Points of Each Compound Isolated Figure 8 - FTIR Scan of Pure Acetylsalicylic Acid (Aspirin Tabulated Values for Acetylsalicylic Acid FT-IR Scan LINK

28 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Robert Allara, academic specialist Shahrzad Taghdissi, and Ms. Beata Mourad for aiding in the editing process. Contact Information Dr. Vikul Rajpara: [email protected] Professor Sherry Heidary: [email protected] Dr. Mushtaq Khan: [email protected] Nathaniel Baptista [email protected] Lucas Chudoba [email protected] Hayley Crowell [email protected] Abigail Dizon [email protected] Carine Medellus [email protected] Krystal Villalobos [email protected] References Ashenhurst, J. Infrared spectroscopy: A quick primer on interpreting spectra. Master Organic Chemistry. https://www.masterorganicchemistry.com/2016/11/23/quick_analysis_of_ir_spectra/ (accessed 2023-01-20). PubChem. Methyl Salicylate. Nih.gov. https://pubchem.ncbi.nlm.nih.gov/compound/Methyl-salicylate (accessed 2023-01-20). EBSCO Information Services. https://web.s.ebscohost.com/ehost/pdfviewer/pdfviewer?vid=0 (accessed 2023-01-20). FTIR Apodization: Data Collection, 2015. https://www.thermofisher.com/us/en/home/indus Chemistry 211 Experiment3. Miracosta.edu Nih.gov.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317/(Accessed 2023-01-20trial/spectroscopy-elemental-isotope-analysis/spectroscopyelemental-isotope-analysis-learning-center/molecular-spectroscopy-information/ftir-information/ftir-basics.html. (Accessed 2023-01-20). Illustrated Glossary of Organic Chemistry - Fingerprint region. Ucla.edu. http://www.chem.ucla.edu/~harding/IGOC/F/fingerprint_region.html(accessed 2023-01-20). IR table. Ucla.edu. https://www.chem.ucla.edu/~bacher/General/30BL/IR/ir.html (accessed 2023-01-20). Jack DB, One hundred years of Aspirin. The Lancet 1997:350: 437-39 The story of Aspirin. Aspirin Foundation. https://www.Aspirin-foundation.com/history/the-Aspirin-story/ (accessed 2023-01-20). Jack DB. Lepodise, L. M. Low-Frequency Terahertz Spectrum of Acetylsalicylic Acid over a Wide Temperature Range Investigated by FTIR Spectroscopy. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2019, 217, 35–38. https://doi.org/10.1016/j.saa.2019.03.068. (Accessed 2023-01-20). PubChem. Methyl Salicylate. Nih.gov. https://pubchem.ncbi.nlm.nih.gov/compound/Methyl-salicylate (accessed 2023-01-20). Ye, Q.; Spencer, P. Analyses of Material-Tissue Interfaces by Fourier Transform Infrared, Raman Spectroscopy, and Chemometrics. In Material-Tissue Interfacial Phenomena; Spencer, P., Misra, A., Eds.; Elsevier, 2017; pp 231–251 Pablo. Chemical Properties of Aspirin and its Effect on the Body. Noah Chemicals. https://noahchemicals.com/blog/chemical-properties-ofAspirin-and-its-effect-on-the-body/(accessed 2023-01-20).

29 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 The Effect of Thyme Oil on the Antimicrobial Properties of Ethylene Vinyl Acetate (EVA) Plastic Anam Khan1 , Montserrat Ramon1, 2, Yamanni Tay1, 2, Faiza Fayyaz1 , Christian Traba3 and Clive Li1 School of STEM, Hudson County Community College, Jersey City, NJ Jose Marti STEM Academy, Union City, NJ 07087 Department of Chemistry, Biochemistry, and Physics, Fairleigh Dickinson University, Teaneck, NJ 07666 Abstract- The rising concern over bacterial contamination and the persistence of antibiotic-resistant strains has necessitated innovative approaches to combat microbial pathogens. This study explores the potential of essential oils, particularly thyme oil, as a natural antimicrobial agent for enhancing the efficacy of plastics in inhibiting the growth of two prominent bacterial species, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). To ascertain the antimicrobial properties of essential oils, we initially tested eight distinct essential oils of —Lemongrass, Wintergreen, Peppermint, Clove, Citronella, Cedarwood, Thyme and Geranium against E. coli and S. aureus. To achieve this, we employed the Kirby Bauer Method, a standardized and widely accepted assay for assessing the antimicrobial properties of materials. Our results revealed that thyme oil exhibited the most robust antimicrobial activity, effectively inhibiting the growth of both E. coli and S. aureus. This finding served as a basis for further exploration into its potential application in plastic materials. Subsequently, we incorporated thyme oil into Ethylene Vinyl Acetate (EVA) plastic matrices to create antimicrobial plastic composites. We tested the antimicrobial activity of the composite and found that the EVA/thyme oil composite exhibited remarkable efficacy in inhibiting the growth of E. coli and S. aureus. The zones of inhibition observed around the thyme oil-infused plastic samples were indicative of the significant antimicrobial activity of these materials. The inhibition zones were notably larger when compared to control samples devoid of thyme oil. Introduction In recent years, the search for effective antimicrobial agents has intensified due to the escalating global crisis of antimicrobial resistance. Essential oils, particularly those with volatile aromatic compounds extracted from plants, have gained significant attention due to their diverse and potent antimicrobial properties. Hammer et al (1999). used the agar dilution method to investigate the antibacterial properties of 52 types of essential oils and plant extracts against Acinetobacter baumanii, Candida albicans, Enterococcus faecalis, Escherichia coli (E. coli), Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhimurium, Serratia marcescens and Staphylococcus aureus (S. aureus). They found lemongrass, oregano and bay oil inhibited all organisms at concentrations of <2.0% (v/v). They also used the broth microdilution method to investigate the antibacterial properties of 20 plant oils and extracts against C. albicans, S. aureus and E. coli. and found that the lowest minimum inhibitory concentrations were 0.03% (v/v) thyme oil against C. albicans and E. coli and 0.008% (v/v) vetiver oil against S. aureus. (Hammer et al., 1999) Bakkali et al. describes the chemical compositions in essential oils and their biological effects (Bakkali et al., 2007). These antimicrobial properties of the essential oil have opened avenues for creating antimicrobial plastics, although their integration is not straightforward due to immiscibility and phase separation challenges. Researchers have delved into innovative methods to overcome this hurdle, leading to groundbreaking studies. For instance, a study conducted by Wang et al. demonstrated a novel approach where oregano essential oils (OEO) were infused into starch. Starch, acting as an intermediary, effectively absorbed the oils, creating a stable mixture. Subsequently, this oil-soaked starch was seamlessly incorporated into polyvinyl alcohol (PVA) plastic film. The PVA plastic film with the OEO showed antimicrobial activity (Wang et al, 2022). Additionally, research by Wongphan et al.

30 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 investigated the incorporation of Lesser galangal essential oil into PLA/PBS blend films. They found that the enhanced antimicrobial properties of the film can extend the shelf-life of the extension packaging of cooked rice (Wongphan et al., 2023). In the present study, the antimicrobial properties of eight essential oils, Lemongrass, Wintergreen, Peppermint, Clove, Citronella, Cedarwood, Thyme, and Geranium were investigated against E. coli and S. aureus. The essential oil with the most robust antimicrobial activity was then infused into Ethylene Vinyl Acetate (EVA) plastic matrices to create antimicrobial plastic composites. The antimicrobial activity of the composite was then tested against E. coli and S. aureus. Materials and Methods Essential Oils There were eight different types of essential oils utilized in the disc diffusion tests and the integration into plastic: 1. Lemongrass supplied by Nature’s Oil, 2. Wintergreen supplied by Sun Essential Oil, 3. Peppermint supplied by Nature’s Oil, 4. Clove Oil supplied by Ashwin Pharma, 5. Citronella supplied by Nature’s Alchemy, 6. Cedarwood supplied by Nature’s Oil, 7. White Thyme Oil supplied by Nature’s Oil, and 8. Geranium supplied by Nature’s Alchemy. The essential oils are listed in Table 1. Bacterial Strains and Agar Cultures of E. coli – ATCC - 25922; LOT - 335-575-4 – were used in both the disc diffusion test as well as the evaluation of the plastic beads test. The S. aureus –ATCC 25923; LOT - 360-575- 4 – was also used in the same tests. Muller Hinton Agar was used as the standard for the Kirby Bauer method. Other Plastic beads, EDI-240 Ethylene Vinyl Acetate Copolymer (EVA), were purchased by supplier: Muehlstein, manufacturer: Certene. During the integration process of the oil, a handmade tumbler was used through the making of 3D parts, rotating rotator, an Arduino board with previously made coding, and a USB-B heard piece to connect to an electrical outlet. During this process, a small glass bottle was also used to place the treated beads, covering the cap section with parafilm. Essential Oils Analysis To determine which essential oil contains the most effective antibacterial properties, aliquots of 20 g from each of the eight oils were spotted onto 5 mm filter paper discs and allowed to air dry for 40 min. The discs were then placed onto the surface of Muller Hinton agar (MHA) previously split into 4 quadrants that had been streaked with a suspension of each strain of S. aureus (direct contact) with a cotton swab. Another set of streaked plates was exposed to the E. coli strain (also split into quadrants of 4) by placing an impregnated disc in the center of each petri dish. All plates were incubated at 37 °C for 24-hour periods and zones of inhibition were measured on the agar surface in centimeters the following day. The plates were then placed in a cooled refrigerated environment to prevent the growth of bacteria and data was saved to take pictures shown in Figure 1. The results of the test demonstrated that the thyme oil was the most effective and was chosen to be used as the oil infused into plastic. Infusion of Oils into Plastic To infuse the oil into the plastic, a Handmade Drum Tumbler was used from Hudson County Community College. The Thyme Oil was infused into the plastic beads at room temperature. 22 grams of plastic beads and 6 grams of Thyme oil were weighed and placed into a glass bottle. Parafilm was placed around the rim of the bottle once the cap was closed to avoid oil spillage and to contain the strong smell in the bottle. The bottle was drum-tumbled at 20 RPM for 3 days. After the tumbling, the oil was soaked into the plastic beads and there were no visual oil droplets on the plastic bead surface. Evaluation of Treated Plastic The bacterial strains were obtained through the manufacturer and placed in a cooled environment until needed. Utilizing the Kirby Bauer Method, agar plates were prepared. Plastic beads with or without infused Thyme oil are placed in bacteria-streaked agar plates, as listed in Table 2.

31 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Once the plastic beads were placed into agar plates and labeled respectively, they were incubated at 37 °C for 24-hour periods, and zones of inhibition measured on the agar surface in centimeters the following day. Results and Discussion The results demonstrate the antimicrobial properties of thyme oil and its successful implementation into plastic beads. During the disc diffusion test, shown in Figure 1, the thyme oil was able to create the greatest zone of inhibition by exceeding the quadrant it was placed into. Thus, this allowed for the decision to be implemented into plastic to be made. The images shown in Figure 2 and Figure 3 show that the thyme oil and its integration into plastic materials proved to be effective against both E. coli and S. aureus. Table 2 is a summary of images shown from Figures 4 and 5 and the data reveal the zone of inhibition. On average the zone of inhibition was 2.3 cm for E. coli which was treated with plastic and 2.2 cm for S. aureus treated with plastic. Figure 1. Visual results of the paper diffusion test of essential oils. 1. Lemongrass, 2. Wintergreen, 3. Peppermint, 4. Clove, 5. Citronella, 6. Cedarwood, 7. White Thyme, and 8. Geranium. Figure 2. Image of E. coli Agar plates next to the control plate for comparison. On the left is a plastic bead infused with thyme oil, and on the right plastic bead without oil (control). Figure 3. Image of S. aureus agar plates next to the control plate for comparison. On the left is a plastic bead infused with thyme oil, and on the right plastic bead without oil (control). Figure 4. Image of E. coli plates with ruler to show the diameter of the zone of inhibition in different duplicate samples.

32 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Figure 5. Image of S. aureus plates with ruler to show the diameter of the zone of inhibition in different duplicate samples. Table 1. Table with essential oils analysis results Table 2. Table highlighting the zone of inhibition of the essential oils of plastic bead test. Conclusion The effectiveness of the oil-infused plastic beads against gram-positive and negative bacteria proves there are better approaches to naturally found inhibition against different strains of bacteria. The application of this plastic can be implemented in various fields, including but not limited to food packaging, where thyme-infused plastics can help extend the shelf life of perishable items. By inhibiting bacterial and fungal growth, medical equipment made from antimicrobial plastic could help reduce the risk of healthcare-associated infection and household products, reducing the need for chemical antimicrobial agents. Contact Information Clive Li: [email protected] Faiza Fayyaz: [email protected] References Bakkali, F.,Averbeck, S., Averbeck, D., & Idaomar, M.(2008). Biological effects of essential oils - a review. Food and Chemical Toxicology, 46(2), 446-475. Hammer, K. A., Carson, C. F., & Riley, T. V. (1999).Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology, 86(6), 985-990. Wang, J., Chen, C., Xie, J. (2022). Loading oregano essential oil into microporous starch to develop starch/polyvinyl alcohol slow-release film towards sustainable active packaging for sea bass (Lateolabrax japonicus) Industrial Crops & Products, 188, 115679 Wongphan,P.,Nampanya,P.,Chakpha,W.,Promhu ad,K.,Laorenza,Y,Leelaphiwat,P.,Bumbudsanpha roke, N., Sodsai, J., Lorenzo, J., Harnkarnsujarit, N. (2023). Lesser galangal (Alpinia officinarum Hance) essential oil incorporated biodegradable PLA/PBS films as shelf-life extension packaging of cooked rice. Food Packaging and Shelf Life, 37, 101077 Essential Oils Visible Zone of Inhibition in E. coli Visible Zone of Inhibition in S. aureus Lemongrass Yes Yes Peppermint No No Cedarwood No No Thyme Oil Greatest Zone Greatest Zone Geranium Yes No Citronella Yes Yes Wintergreen No No Clove Oil Yes No Treatment Zone of Inhibition Control E. coli None E. coli + Plastic NO oil None E. coli + Plastic + oil Average 2.3 cm Control S. aureus None S. aureus + Plastic NO oil None S. aureus + Plastic + oil Average 2.2 cm

33 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Did Napoleon’s Brother, the King of Spain, Grow Trees in Bordentown New Jersey from Seeds Collected by Lewis and Clark? Zachary Farawell, Mentor: Associate Professor Jennifer Ebert, M.S.E. STEM Division, UCNJ Union College of Union County, NJ Abstract—Joseph Bonaparte, former King of Spain, and brother of Napoleon Bonaparte, built an estate in Bordentown, New Jersey after Napoleon's defeat. Two large Osage Orange trees are located on this property. Osage Orange trees were first introduced to the eastern U.S. by Lewis and Clark, who sent seed samples to President Thomas Jefferson while on their expedition. Jefferson had nurseries in Philadelphia to grow these seeds and the nurseries began selling the trees soon after. Did Joseph Bonaparte plant two of these Osage Orange trees on his estate? In this study, data were collected to answer this question by comparing Osage Orange trees known to be from samples collected by Lewis and Clark to the Osage Orange trees on Joseph's property. The findings suggest that it is highly plausible that the trees located on Joseph's old estate came from Lewis and Clark samples. Introduction Joseph Bonaparte was the older brother of the infamous Napoleon Bonaparte. After his conquest of Spain, Napoleon made Joseph the King of Spain [4]. After Napoleon’s defeat and exile, Joseph fled Europe in 1815, and sought refuge in the United States. The young nation was grateful to the French for their aid against the British during the revolution, and so, welcomed the deposed monarch with open arms. Joseph's wife would not accompany him to America during his 23-year stay [3]. Joseph searched many parts of the U.S. for a suitable place to settle down, and eventually fell in love with a site on the Delaware River in Bordentown, New Jersey. New Jersey state law at the time forbade foreigners from purchasing land, so Joseph purchased the property through a friend in 1816 and named his new estate Point Breeze [3]. a b Figure 1. (a) Joseph Bonaparte (b) Napoleon Bonaparte Figure 2. Map of Point Breeze, Bordentown NJ

34 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Point Breeze was initially 211 acres, but after New Jersey repealed the law preventing foreigners from buying land, Joseph bought 1000 more acres of surrounding land. Joseph became an avid gardener and covered his new estate in exotic and beautiful plants to show the gardens to the American elite [3]. Many of his guests included U.S. presidents, politicians, artists, inventors, and even royalty. In 1820, the house at Point Breeze caught fire, and was replaced with a mansion at a different site on the property [2]. Joseph was well-liked in Bordentown and was known for his philanthropy. He paid for many infrastructural projects and employed many of the residents [3]. Joseph would remain at Point Breeze until 1832 when he left for England to try to seat his nephew on the throne of France [2]. He would travel between America and various parts of Europe until he died in 1844 [2]. After his death, Point Breeze was inherited by his grandson who sold it in 1847. The property eventually ended up in the hands of the British Government for their consulate in the United States. They tore down Joseph’s mansion in an act of spite and replaced it with an Italian-style villa [3]. In the 20th century, Point Breeze was acquired by a Catholic Missionary group [3], who would later sell it to the town of Bordentown. Today, Point Breeze is a public park and the site of the City of Bordentown municipal building. Figure 3. Map of Point Breeze, Bordentown, NJ Joseph Bonaparte’s Garden at Point Breeze was renowned for its beauty and variety. The garden covered several acres of land and was divided into different sections, each with its own distinct character [3]. One of the most impressive features of the garden was a large ornamental lake, which was stocked with exotic fish and surrounded by flowering trees and shrubs. The lake was also home to several swans and other waterfowl, which added to its picturesque charm. Other sections of the garden included a fruit orchard, a vegetable garden, and a collection of rare and exotic plants from around the world. Joseph was known for his passion for botany and horticulture, and he often experimented with new varieties of plants and flowers. In addition to its natural beauty, the garden at Point Breeze was also home to many works of art, including statues, fountains, and ornamental buildings. Joseph was a collector and patron of the arts, and he used his garden as a showcase for many of his most prized possessions [3]. Interestingly, there are two Osage Orange trees in a central location of the estate [Figure 1]. These trees are not native to the eastern seaboard and were not widely available in the early 19th century [1]. They were first introduced to the area by Lewis and Clark when they sent cuttings and seeds of Osage Orange trees to the land east of the Mississippi River (1804 and 1806). President Thomas Jefferson, who sent Lewis and Clark on their expedition, was the one who received the samples they sent eastward [1]. Jefferson gave the cuttings and seeds to Philadelphia-based nursery owner Bernard McMahon, and then McMahon planted some seeds and cuttings to experiment with them and to start selling them [5]. The trees that McMahon planted to test the seeds remain on the property of St. Peter’s Episcopal Church in Philadelphia [Figure 2]. Soon after McMahon successfully started growing Osage Oranges, Landreth’s Seed and Nursery, also in Philadelphia, began to sell Osage Orange trees [2]. Joseph Bonaparte bought many trees from Landreth in 1821, but the receipt was very minimal in its description of what was sold. The location of the trees, Joseph’s love of exotic plants, the close connection

35 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 to the specific vendors selling the trees, and the prestige tied to owning a rare tree from a dangerous expedition leads to an interesting question. Did Joseph Bonaparte plant a tree collected by Lewis and Clark on their expedition on his property in Bordentown? To answer this question, this study compared the two Osage Orange trees at Point Breeze with Osage Orange trees of known ages in Philadelphia and Staten Island, New York. Osage Orange trees [Maclura pomifera] are deciduous trees native to northeastern Texas, southwestern Arkansas, and southeastern Oklahoma [7]. They typically grow between 20 to 50 feet tall with trunks 1.5 to 3 feet in diameter. But given the ideal conditions, they can grow to be much larger. They are known for their thick and rough bark that has a slightly orange hue. Their young branches are thorny, and if planted close together, they grow more like hedges than trees. Osage Orange trees produce large green fruit often referred to as hedge apples. They are hardy trees and can grow in most parts of the U.S., except for extremely cold regions such as the New England region. They tolerate various kinds of soil and can survive in wet and dry conditions. Male trees contain flowers that produce pollen, while female trees, once pollinated, bear fruit. The fruit can be eaten by a variety of animals, who will then defecate the seeds, allowing for new trees to sprout. Osage Orange trees can also be found in the western part of the U.S. Native Americans planted Osage Orange trees as they valued the trees for their hardwood. Various parts of the tree were used to create bows, medicine, dyes, and insect repellants. When European settlers came in contact with the trees, they primarily used them as natural fences, and the wood as fence posts [7]. Materials and Methods The data in this study was collected by measuring the Osage Orange trees under study at Point Breeze, as well as the four trees at St. Peter’s Episcopal Church in Philadelphia, and the lone tree at the Olmstead-Beil House on Staten Island, New York. Measurements were taken by using a measuring tape to obtain the trunk circumference of the trees at 4.5 feet above the ground and measuring the height of the tree using a Spiegel Relaskop. The average crown spread was determined by measuring the distance of the furthest spread, and the shortest spread and calculating the average. The three measurements gathered for each tree were converted into points based on the point system developed by the USDA. The equation to do so is C+H+(0.25S), where C is the trunk circumference in inches, H, is height in feet, and S is the average crown spread in feet [6]. Results and discussion The resulting measurements directly taken from the trees, and the points derived from those measurements are shown in Table 1. In Table 2, trunk diameter, the ages of the trees, and annual trunk growth are displayed. The diameters were calculated from the measured trunk circumferences. The ages of all the trees are known except for the ages of the trees at Point Breeze, for which an assumed age of 216 years old was used. Finally, the annual trunk growth was calculated from the diameters of the trunks and the ages of the trees, Figure 4. Osage Orange Trees at Point Breeze

36 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Figure 5. Osage Orange Tree #4 at St. Peter’s Episcopal Church in Philadelphia Table 1: A Comparison of Trunk Circumference, Height, Crown Spread, and total Points. Table 2: A Comparison of Annual Trunk Diameter Growth Conclusion The purpose of this study was to investigate possible connections between the two Osage Orange trees at Point Breeze and the samples known to be obtained by Lewis and Clark. While there is no prior literature on this specific topic, there are many writings on Joseph Bonaparte, Point Breeze, Lewis and Clark, and the introduction of Osage Orange trees to the eastern United States. The presence of large Osage Orange trees on the former property of Joseph Bonaparte raises questions about their origins, and the written sources mentioned above make it highly plausible that Joseph Bonaparte procured these trees from samples sent by Lewis and Clark. From this, we have developed the hypothesis that these trees are from the Lewis and Clark expedition. The analysis of the data, presented in Table 1 and Table 2, indicates that the trees at Point Breeze are similar in size to the trees in Philadelphia. Table 1 demonstrates that the trees at Point Breeze are firmly within the point range of the trees of known ages. Note the double-trunk tree is slightly smaller as the tree's energy and resources were less centralized when compared to the single-trunk trees. Furthermore, Table 2 shows that the trunk diameters of both trees at Point Breeze are firmly within the diameter range of the trees at Philadelphia. Also, in Table 2, the annual growth in trunk diameter indicates that both of the trees at Point Breeze fit well within the range of annual trunk growth of the trees located in Philadelphia. For the annual trunk growth of the trees at Point Breeze it was assumed that the trees were 216 years old for the hypothesis. The similar sizes of these trees support the plausibility of the hypothesis; however, without written documentation to prove who bought the trees, and from where they came one cannot definitively prove their origins. These findings support the hypothesis that Joseph purchased these trees in the early 19th century from samples collected by Lewis and Clark. The fact that Lewis and Clark specifically sent Osage Orange tree samples to Philadelphia, and Joseph frequently purchased plants from nurseries in that city, further supports this argument. Given

37 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6| No. 1 | Fall 2023 Joseph's love for exotic plants and his desire to impress his wealthy guests, it seems likely that he would have taken the opportunity to display a new and rare species of tree. While there is no written evidence of Joseph's specific purchase of Osage Orange Trees, the available evidence makes a strong case that these trees originated from the Lewis and Clark samples. However, without definitive proof of the origin of the trees, their exact provenance cannot be established. What would give definitive answers to the true age of the trees at Point Breeze would be to cut the trees down and count the annual tree trunk rings or coring the trees with a forester’s increment borer. However, it wasn’t possible to obtain permission to core the trees for this study, nor is it reasonable to destroy the trees to obtain their ages. Acknowledgment This research was made possible by the National Science Foundation, IRAP grant 1832425. Its contents are solely the responsibility of the award recipient and do not necessarily represent the official views of the National Science Foundation. The author wishes to acknowledge the mentorship and guidance of Senior Professor Thomas Ombrello, PhD and UCNJ Union College of Union County, NJ STEM Division for their continuous support. Contact Information Professor Jennifer Ebert: [email protected] Zachary Farawell: [email protected] References 1- Kris. “The Osage Orange - Discover Lewis and Clark.” Discover Lewis & Clark, 5 Dec. 2022, lewis-clark.org/sciences/plants/osage-orange. 2- Stroud, Patricia. “The Man Who Had Been King, the American Exile of Napoleon’s Brother Joseph.” (2005). University of Pennsylvania Press. 3- Walton, Geri. “Point Breeze Estate or Joseph Bonaparte’s Park - Geri Walton.” Geri Walton,(Nov.2019) www.geriwalton.com/point-breeze-estate-orjoseph-bonapartes-park 4- Encyclopaedia Britannica. “Joseph Bonaparte | King of Spain and Naples.”Encyclopedia Britannica, 11 Feb. 2023, www.britannica.com/biography/JosephBonaparte. 5- Philadelphia.” Monticello” www.monticello.org/site/research-andcollections/philadelphia. Point Breeze - History of Early American Landscape Design https://heald.nga.gov/mediawiki/index.php/P oint-Breeze 6- “Howmto Measure a Big Tree.” http://www.fs.usda.gov/Internet/FSE_DOCU MENTS/stelprdb5202838.pdf#:~:text=Each %20tree%20you%20nominate%20will%20b e%20scored%20using,is%20described%20in %20the%20re mainder%20of%20this%20document, Accessed 5 Apr. 2023. 7- “USDA Plant Guide, Osage Orange.” USDA Natural Resources Conservation Service, https://plants.usda.gov/DocumentLibrary/plantguide/pdf/pg_mapo.pdf#:~:text=Osage%20o range%20%20s%20a%20pioneering%20species%20forever%20invading,species%2C%20state%20noxious%20%20status%2C%20and%20wetland%20indicator%20values%29.

2 UCNJ Union College of Union County, NJ | Undergraduate Research Journal Volume 6 | No. 1 | Fall 2023 Union County Board of County Commissioners Sergio Granados Commissioner – Chairman Kimberly Palmieri-Mouded Commissioner – Vice Chair James E. Baker, Jr. Commissioner Joseph C. Bodek Commissioner Dr. Angela R. Garretson Commissioner Bette Jane Kowalski Commissioner Lourdes M. Leon Commissioner Alexander Mirabella Commissioner Rebecca L. Williams Commissioner UCNJ does not discriminate and prohibits discrimination, as required by state and/or federal law, in all programs and activities, including employment and access to its career and technical programs.