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Sabnis Uta 2502D 12838.Pdf (5.069Mb) INVESTIGATION OF HOW MICROBES INVOLVED IN ANEROBIC DIGESTION OF VINASSE CHANGE AS FUNCTIONS OF TEMPERATURE, VINASSE COMPOSITIONS AND TIME by MADHU SANJOG SABNIS Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON August 2014 Copyright © by Madhu Sanjog Sabnis 2014 All Rights Reserved ii Acknowledgements I would like to express my wholehearted gratitude to my advisor Dr. Melanie Sattler and Dr. Jorge Rodrigues who has always been an invaluable guide and academic mentor. I really appreciate Dr. Melanie Sattler for her encouragement, support, and advice throughout my research work. I cannot express my gratitude to Dr. Rodrigues for providing excellent ideas and advice that propelled my research. The supervising committee members Dr. James Grover, Dr. Andrew Hunt, and Dr. Sahadat Hossain, are most appreciated and gratefully acknowledged for their valuable suggestion towards this study. A special and personal acknowledgement is due to the Dr. Melanie Sattler; for her unrestricted personal guidance throughout this study, for bringing out the best out of my ability. I would like to thank Mr. Paul Shover for his tremendous help with my experimental set-up work. I also thank Shammi Rahman for helping me throughout my research. I would like to thank Victor Pylro and Jill Castoe who helped me during my experimental analysis and sequencing process. Last but not the least; I thank God and my loving family - Mom, Dad, brother, sister, and my family-in-law. They are my real strength and have always believed in my abilities to not only dream but strive to make those dreams a concrete reality. I'm greatly indebted to my brother for his ceaseless encouragement and blessings, without which I wouldn't have committed myself to pursue higher studies. Finally, I would like to mention my special thank to my husband Sanjog Sabnis for his continual help, love and support to accomplish my goal. July 18, 2014 iii Abstract INVESTIGATION OF HOW MICROBES INVOLVED IN ANEROBIC DIGESTION OF VINASSE CHANGE AS FUNCTIONS OF TEMPERATURE, VINASSE COMBINATIONS AND TIME MADHU SANJOG SABNIS, PhD The University of Texas at Arlington, 2014 Supervising Professor: Melanie Sattler Recent fossil fuel supply crunches and price spikes have prompted increased interest in ethanol as an alternative transportation fuel. However, vinasse, the liquid residue left from the distillation of ethanol, poses serious disposal challenges due to its high acidity, chemical oxygen demand, and concentrations of solids, nitrogen, phosphorous, and potassium. On average, 12-14 liters of vinasse are produced per liter of ethanol. Brazilian production of ethanol is estimated as 15.1 billion liters per year, meaning that the vinasse treatment and disposal problem for Brazil is particularly significant. Degradation via anaerobic microbes has been demonstrated as a viable option for vinasse treatment, which also produces renewable energy in the form of methane as a byproduct. Methane and other gases, produced in the anaerobic digestion of vinasse, are enough to generate 3.6 to 10.60 megawatts of electricity when vinasse BOD ranges from 1.06 to 3.12 lb/cu ft. The objective of this research was to investigate the microbes involved in the various stages of anaerobic degradation of vinasse, and how the numbers and species of iv microbes varied as functions of time, vinasse composition, and reactor temperature. This information will be useful for future seeding of industrial-scale vinasse treatment processes with appropriate microbes. Vinasse from production of ethanol from corn and milo was obtained from two U.S. companies; additional synthetic vinasse with various concentrations of parameters (COD, N, P, K, S) was prepared in the laboratory. Vinasse was placed in 6.8L glass bioreactors seeded with 10% sewage sludge obtained from an anaerobic process at a wastewater treatment plant. The reactors were operated at three mesophilic temperatures (30, 35 & 40˚C) in batch mode for 15-89 days, depending on methane production. Three molecular techniques, DNA extraction PCR, and Miseq, were used to determine microbial communities in each reactor as a function of time. Post-sequencing microbial community analysis was performed using QIIME software. As temperature increased, the run time generally decreased, as expected. Overall, the synthetic vinasse combinations had higher percentages of Archaea in the runs compared to real vinasse compositions. The synthetic vinasse thus produced higher levels of methane compared to the real vinasse combinations. The synthetic and real vinasse combinations at 30, 35, and 40°C contain higher percentages of Bacteroidetes, Firmicutes, and Proteobacteria and lower percentages of Archaea, Acidobacteria, Tenericutes, Spirochaetes, Synergistetes, Chloroflexi, Lentisphaerae, WWE1, Verrucomicrobia, Tenericutes, Spirochaetes, Synergistetes, and Plantomycetes. There were no overall trends of microbial phyla observed with time. v Table of Contents Acknowledgements .............................................................................................................iii Abstract .............................................................................................................................. iv List of Illustrations ............................................................................................................... x List of Tables ................................................................................................................... xviii Chapter 1 Introduction......................................................................................................... 1 1.1 Background ............................................................................................................ 1 1.2 Research Objective ................................................................................................ 2 1.3 Dissertation Organization ....................................................................................... 2 Chapter 2 Literature Review ............................................................................................... 4 2.1 Background on Vinasse .......................................................................................... 4 2.2 Anaerobic Process Basics .................................................................................... 13 2.3 Factors Affecting Anaerobic Processes ............................................................... 14 2.3.1 Temperature................................................................................................ 14 2.3.2 pH ................................................................................................................ 15 2.3.3 Nutrient Content .......................................................................................... 16 2.3.4 Toxic Substance Concentrations ................................................................ 16 2.3.5 Batch or Continuous Operation................................................................... 16 2.3.6 Reactor Mixing ............................................................................................ 17 2.4 Methods for Microbe Determination ..................................................................... 18 2.5 Previous Studies of Microbial Communities in Anaerobic Bioreactors ................ 28 2.6 Factors Affecting Anaerobic Digestion and Biogas Production ............................ 32 Chapter 3 Materials & Methods ........................................................................................ 38 3.1 Introduction ........................................................................................................... 38 3.2 Task 1 Experimental Design................................................................................. 39 vi 3.2.1 Temperature................................................................................................ 41 3.3 Task 2 Setting Up Laboratory-Scale Reactors .............................................. 41 3.3.1 Vinasse collection ....................................................................................... 41 3.3.2 Synthetic Vinasse ....................................................................................... 42 3.3.3 Real Vinasse ............................................................................................... 43 3.3.4 Reactors ...................................................................................................... 44 3.4 Task 3 Operating and Monitoring Laboratory Scale Bioreactors ......................... 46 3.4.1 Gas Measurement ...................................................................................... 46 3.4.2 Monitoring of pH, Temperature and Gas Fitting Connections .................... 47 3.5 Task 4 Sample Collection ..................................................................................... 48 3.5.1 Septa ........................................................................................................... 48 3.5.2 Syringes and Needles ................................................................................. 49 3.5.3 Gas Sampling Bag ...................................................................................... 49 3.5.4 Centrifuge Tubes for Sample Storage ........................................................ 50 3.5.5 Techniques for Microbe Determination ......................................................
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