West Chester University Digital Commons @ West Chester University West Chester University Master’s Theses Masters Theses and Doctoral Projects Spring 2020 Microbial Energy Storage and Antimicrobial Effects of Metal Nanoparticles Octavia Allen [email protected] Follow this and additional works at: https://digitalcommons.wcupa.edu/all_theses Part of the Biology Commons Recommended Citation Allen, Octavia, "Microbial Energy Storage and Antimicrobial Effects of Metal Nanoparticles" (2020). West Chester University Master’s Theses. 101. https://digitalcommons.wcupa.edu/all_theses/101 This Thesis is brought to you for free and open access by the Masters Theses and Doctoral Projects at Digital Commons @ West Chester University. It has been accepted for inclusion in West Chester University Master’s Theses by an authorized administrator of Digital Commons @ West Chester University. For more information, please contact [email protected]. Microbial Energy Storage and Antimicrobial Effects of Metal Nanoparticles A Thesis Presented to the Faculty of the Department of Biology West Chester University West Chester, Pennsylvania In Partial Fulfillment of the Requirements for the Degree of Master of Science By Octavia A. Allen May 2020 © Copyright 2020 Octavia A. Allen All Rights Reserved Acknowledgements There are a number of individuals I would like to acknowledge and thank for their role in helping me complete my thesis work. I would like to thank the faculty and students of West Chester University’s Biology Department for their encouraging words while I was working on this project for the past 2 years. Also, to Enrique Mentado and Jim Wheeler, thank you for assisting me with learning new lab techniques and how to utilize certain lab instruments. Jim, thank you so much for your time and lending your strong chemistry background knowledge for the benefit of this project. To Samantha Shumlas, thank you for allowing me to use the Center for Microanalysis and Imaging Research and Training (CMIRT) Facility and helping with obtaining results from using X-ray Diffraction, Energy-Dispersive X-ray Spectroscopy, and Scanning Electron Microscopy. To my committee members, Dr. Pisciotta, Dr. Mbuy, and Dr. Pagán, thank you for all your help, advice, and patience. All of you have been so helpful in not only the completion of this project, but also great mentors for anything I had questions about. Thank you for teaching me so much about the field of microbiology, bioenergy, and being there for me every single step of the way Dr. Pisciotta. I truly appreciate it; you are an awesome advisor! Lastly, but certainly not least, I would like to thank God. It was God who blessed me with the strength, determination, ambition, patience, and focus to complete this thesis project. I literally would not have been able to successfully complete this work without Him. Abstract Microbial Energy Storage and Antimicrobial Effects of Metal Nanoparticles By Octavia A. Allen Chairperson: John Pisciotta, PhD With the use of Microbial Electrolysis Cells (MECs) and anaerobic digester (AD) designs, wastewater treatment could be more effectively processed. Inoculation of anaerobic microorganisms, methanogens specifically, can help convert organic matter in wastewater metabolizing it to produce CH4. Most MECs are stationary and are not actively mixed to promote expedited CH4 production. The objective of this work was to design Wind-Actuated Vibrating Electrochemical (WAVE) digesters and utilize electrical current to increase CH4 production rates generated by anaerobic microorganisms in MECs, optimizing efficiency of wastewater treatment. It was hypothesized WAVE mixed digesters will enhance conversion of wastewater into biogas compared with conventional AD designs, and faster startup of biogas production will occur in WAVE actuated digesters vs controls. Also, higher voltage of 900 mV will result in greater biogas production compared to 700 mV or conventional AD (i.e. disconnected). Current was applied at 700 mV and 900 mV to specified reactors in coordination with vertical and horizontal WAVE digester designs. Concluding experimentation, there was no significant difference between voltage or WAVE digester design on CH4 production concluding the 8-week study. Compared to the stationary, traditional anaerobic digester, none of the 700 mV applied MECs, despite WAVE digester design, had a significantly higher CH4 accumulation after 8 weeks. Compared to the stationary, traditional anaerobic digester, none of the 900 mV applied MECs, despite WAVE digester design, had a significantly higher CH4 accumulation after 8 weeks Compared to traditional anaerobic digesters, CH4 recovery while treating wastewater is optimized in digesters with an applied voltage. Keywords: Microbial Electrolysis Cells, anaerobic digester, wastewater, microorganisms Table of Contents Introduction ................................................................................................................................. 1 Wastewater Treatment .................................................................................................... 1 Anaerobic Digestion ....................................................................................................... 2 Bioelectrochemical Systems (BESs)............................................................................... 4 Microbial Electrolysis Cells (MECs) .............................................................................. 6 Modifications of BESs for Methane Production Enhancement ...................................... 7 Materials and Methods ................................................................................................................ 9 Microbial Energy Storage ............................................................................................... 9 WAVE Digester Design ...................................................................................... 9 Construction of WAVE Digesters .......................................................... 9 Inoculation ........................................................................... ………….10 Quantification of Electrogenic Activity ............................................................ 10 Gas Chromatography ............................................................................ 10 Chronoamperometry ............................................................................. 11 Microbial Community Analysis ........................................................................ 11 DNA Extraction .................................................................................... 11 DNA Quantification .............................................................................. 12 16S rDNA PCR Amplification ............................................................. 12 Denaturing Gradient Gel Electrophoresis (DGGE) .............................. 13 Nanopore DNA Sequencing ................................................................. 14 Antimicrobial Effects of Metal Nanoparticles……….………………………………..15 Antimicrobial Metals ........................................................................................ 15 Waterborne Viruses and Indicator Species ....................................................... 15 Plaque Assay Procedure ........................................................................ 16 Effects of Various Metals on Gas Production Rates ......................................... 16 Effects of NaCl Concentration on Gas Production Rates ................................. 17 Antimicrobial Efficacy of Nanoparticles using 96 Well High Throughput ...... 17 Chemical Makeup and Nanostructure of Nanoparticles ................................... 18 Results…………………………..……………………………………………………………..20 Discussion……………………………………………………………………………………..32 References……………………………………………………………………………………..37 Appendix……………...…………………………………………………….…………………41 List of Tables Table 1 – EDS Weight Percentage of Each Detected Element ............................................... 30 List of Figures Figure 1 – Weekly Average CH4 Accumulated ...................................................................... 20 Figure 2 – Total Average CH4 Accumulated .......................................................................... 21 Figure 3 – DGGE of DNA Samples in WAVE Digester Reactors ......................................... 22 Figure 4 – Identified Microbial Species .................................................................................. 22 Figure 5 – Phylogenetic Linkage of Identified Microorganisms ............................................. 23 Figure 6 – Effect of Fe Anode Cu Cathode vs. T4 Bacteriophage .......................................... 24 Figure 7 – Effect of Anode Metal on Gas Production ............................................................. 25 Figure 8 – NaCl Concentration vs Gas Production at 15 V .................................................... 26 Figure 9 – Fe Anode 4 Hours .................................................................................................. 27 Figure 10 – Zn Anode 4 Hours ................................................................................................ 27 Figure 11 – Fe Anode 24 Hours .............................................................................................. 28 Figure 12 – Zn Anode 24 Hours .............................................................................................. 28 Figure 13 – SEM Image of Washed Zn Nanoparticles ...........................................................