POTENTIAL APPLICATION AND PERFORMANCE OF METHANOTROPHIC MIXED CULTURE UNDER MAIN STREAM CONDITION IN WASTEWATER TREATMENT PLANT Safayat Hosen Suhad A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE GRADUATE PROGRAM IN CIVIL ENGINEERING York University Toronto, Ontario December 2019 ©Safayat Hosen Suhad, 2019 ABSTRACT Wastewater treatment plants (WWTP) produce significant amount of biogas during sludge digestion in anerobic digestion (AD). Biogas is mainly methane gas which is a potent greenhouse gas (GHG) causing global warming. Direct use of biogas as fuel is an unattractive option due to its low efficiency, storage and transportation difficulty. There is an attractive option to convert methane gas biologically into more desirable forms like methanol and bio polymer. It has been proven through researches that the bio-conversion of methane can be done with the help of a biocatalyst called methanotrophs with higher efficiency and requires no or less energy input. The application of methanotrophs into WWTP has been challenged by its capability to grow under main stream conditions with Chemical Oxygen Demand (COD) along with diverse microbial community. In this study the growing capability of methanotrophic mixed culture was examined under varying COD in synthetic feed. The experiment was done in repeated batch for fifty cycles to obtain the stable behavior of the microbial consortium. In addition, the nitrogen utilization mechanism was investigated to figure out whether the nitrogen is used only for cell synthesis or cell synthesis along with nitrification/ denitrification. The results show that the best microbial performance was attained at COD of 360 mg/L. The average Specific Growth Rate (day-1) was 0.49±0.05, average Biomass Yield (mg-VSS/mg-CH4) was 0.49±0.04 and the average methane uptake rate (mg/hr) was 2.50±0.36. In addition, average COD consumption was 89±5 % at COD 540mg/L and the average ammonia consumption was around 99.72±0.48 %. The percentage of ammonia utilized for cell synthesis was 61-72% and 28-39% of ammonia was converted to a gaseous form of nitrogen. These results demonstrate the feasibility of integrating methanotrophic cultures within the mainstream of existing WWTPs. ii DEDICATION To my beloved late father Md. Abdul Mazid, who is a great inspiration to me to move forward. iii ACKNOWLEDGEMENTS First, I would like to thank the Almighty Allah, for His endless blessings throughout my research work to complete the research successfully. I would like to express my sincere appreciation and gratitude to my thesis supervisor Prof. Ahmed Eldyasti, department of civil engineering, York University, for his continuous support throughout my research work. He is a great mentor, visionary and a good human being. It was a great privilege and honor to work and study under his guidance. I am extremely grateful for what he has offered me. I would also like to thank him for his friendship, empathy, and great sense of humor. I would like to thank my research colleagues specially, Ahmed AlSayed, who mentored me during lab work and provided continuous help during my research. Also, I am thankful to Fergala, Moomen, Danelle, Parin, Parnian, Rana and Sara for their support, suggestions and encouragement. I am grateful to my wife Sharmin Sultana for her continuous support during hard times and tolerating the pain throughout this journey. Without her support it was impossible to finish my thesis. I am glad that our lovely daughter Sareea Safreen was along with us throughout this time. Sareea was a great inspiration to me to finish my master’s degree. Lastly, I am forever indebted to my late father who gave me the encouragement to believe in myself and built my confidence for any future achievements and to my mother who struggled throughout her life to raise me to this position. I am also thankful to my sisters for their support and valuable prayers. iv TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii DEDICATION ............................................................................................................................... iii ACKNOWLEDGEMENTS ........................................................................................................... iv TABLE OF CONTENTS ................................................................................................................ v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix LIST OF ACRONYM..................................................................................................................... x CHAPTER 1: Introduction ............................................................................................................. 1 1.1 Background ........................................................................................................................... 1 1.2 Research Rationale ................................................................................................................ 2 1.3 Research Objective ................................................................................................................ 4 1.4 Thesis Layout ........................................................................................................................ 5 CHAPTER 2: Literature Review .................................................................................................... 6 2.1 Methane Mitigation and Utilization Techniques................................................................... 6 2.1.1 Gas Engines and Turbines .............................................................................................. 6 2.1.2 Biofuel conversion .......................................................................................................... 7 2.1.3 Fuel Cells ...................................................................................................................... 11 2.1.4 Biological methane mitigation...................................................................................... 14 2.2 Methanotrophs ..................................................................................................................... 15 2.2.1 Methanotrophs classification ........................................................................................ 15 2.2.2 Methane assimilation by methanotrophs ...................................................................... 17 2.2.3 Factors affecting methanotrophs growth ...................................................................... 18 2.2.4 pH ................................................................................................................................. 18 2.2.5 Temperature .................................................................................................................. 18 2.2.6 Substrate ....................................................................................................................... 19 2.2.7 Methane to oxygen ratio ............................................................................................... 19 2.2.8 Nitrogen source............................................................................................................. 19 2.2.9 Copper .......................................................................................................................... 20 2.3 Application of methanotrophs in WWTPs .......................................................................... 20 2.3.1 Methanol production by methanotrophs ....................................................................... 21 2.3.2 PHB production mechanism in methanotrophs ............................................................ 24 v 2.3.3 Nitrification and denitrification by methanotrophs ...................................................... 27 2.3.4 Nitrogen removal practice in WWTPs ......................................................................... 29 2.3.5 Integration of methanotrophs in WWTPs ..................................................................... 30 CHAPTER 3: Methanotrophic-heterotrophic mixed culture performance under varying chemical oxygen demand (COD) ................................................................................................................. 33 3.1 Introduction ......................................................................................................................... 33 3.2 Biochemical conversion of methane by methanotrophs ..................................................... 34 3.3 Nitrogen issues in WWTPs ................................................................................................. 34 3.4 Potential application of Methanotrophs in WWTPs ........................................................... 35 3.5 Scope of Works ................................................................................................................... 36 3.6 Materials and methods ........................................................................................................ 36 3.6.1 Operational condition ................................................................................................... 36 3.6.2 Methanotrophs mixed culture enrichment .................................................................... 37 3.6.3 Growth Phase ................................................................................................................ 38 3.6.4 Analytical methods ....................................................................................................... 39 3.7 Results and Discussion ........................................................................................................ 40 3.7.1 Influence of varying COD on methanotrophic microbial activity ................................ 40 3.7.2 Methane, Oxygen and COD consumption by the microbial community ..................... 46 3.7.3 Nitrogen Removal......................................................................................................... 49 3.7.4 Nitrogen Balance .......................................................................................................... 50 3.8 Conclusion........................................................................................................................... 53 CHAPTER 4: Methanol and PHB production by Methanotrophic-heterotrophic mixed culture performance under different COD concentrations ........................................................................ 55 4.1 Introduction ......................................................................................................................... 55 4.2 Materials and Methods ........................................................................................................ 56 4.2.1 Methanol production..................................................................................................... 56 4.2.2 Growth phase ................................................................................................................ 56 4.2.3 Methanol Phase............................................................................................................. 57 4.2.4 PHB production ............................................................................................................ 57 4.3 Results and Discussion ........................................................................................................ 59 4.3.1 Influence of COD on methane and oxygen consumption during methanol production 59 4.3.2 Influence of COD on methane and oxygen consumption during PHB production ...... 60 4.3.3 Influence of COD on methanol and PHB production................................................... 60 vi CHAPTER 5: Conclusion and Future Work ................................................................................. 62 5.1 Conclusion........................................................................................................................... 62 5.2 Future Study ........................................................................................................................ 65 BIBLIOGRAPHY ......................................................................................................................... 66 APPENDIX A ............................................................................................................................... 74 APPENDIX B ............................................................................................................................... 80 vii LIST OF TABLES Table 3.1: Experimental conditions for fed-batch experiments .................................................... 39 Table 3.2: Comparison of different growth parameters at different COD and ammonia concentration ................................................................................................................................. 45 Table 3.3: Average Ammonia consumption, nitrate, nitrite concentration at different COD level ....................................................................................................................................................... 49 Table 3.4: Ammonia utilization pathways at different COD condition ........................................ 53 Table 4. 1: Methanol and PHB production at different COD condition ....................................... 61 viii LIST OF FIGURES Figure 2.1 PHB production pathway in Methanotrophs. (Adapted from AlSayed et al., 2018b) 24 Figure 2.2: nitrogen catabolic pathways in aerobic methanotrophic bacteria .............................. 28 Figure 2.3: Integration of Methanotrophs in to WWTP ............................................................... 31 Figure 3.1: Average Growth rate (d-1), Methane Uptake rate (mg-CH4/hr) and Growth yield (g DCW/gCH4) at COD 0 mg/L, 180 mg/L, 360 mg/L and 540 mg/L for a) first 10 cycles b) final 12 cycles............................................................................................................................................. 44 Figure 3.2: Methane Uptake rate (mg-CH4/hr) at COD: 0 mg/L, 180 mg/L, 360 mg/L and 540 mg/L for a) first 10 cycles b) final 12 cycles ................................................................................ 44 Figure 3.3: Activity of biomass in terms of % CH4 Consumption and % O2 Consumption for a) COD 0 mg/L and ammonia-N 60 mg/L (control), b) COD 180 mg/L and ammonia-N 30 mg/L, c) COD 360 mg/L and ammonia-N 60 mg/L, d) COD 540 mg/L and ammonia-N 90 mg/L ........... 45 Figure 3.4: average percentage of CH4 and O2 consumption for different COD ......................... 46 Figure 3.5: Average COD Consumption as mg/L and percentage at COD 0 mg/L, 180 mg/L, 360 mg/L and 540 mg/L for a) first 10 cycles b) final 12 cycles......................................................... 47 Figure 3.6: Nitrogen balance data at different COD concentration showing In an Out of total nitrogen and amount of nitrogen converted to gas........................................................................ 51 Figure 3. 7: Ammonia utilization pathway showing the percentage of ammonia (in) converted to gas, utilized for cell synthesis and percentage of nitrogen inside the biomass ............................. 52 Figure 4.1: Percentage of CH4 and O2 Consumption during methanol production phase ............ 59 Figure 4.2: Methane and Oxygen consumption Percentage during PHB production ................... 60 ix LIST OF ACRONYM AD : Anaerobic Digester ................................................................................................................ 1 AME-D : Aerobic Methane Oxidation Coupled to Denitrification .............................................. 28 AMO : Ammonia Monooxygenase ............................................................................................... 27 AOB : Ammonia Oxidizing Bacteria .............................................................................................. 2 AS : Activated Sludge ..................................................................................................................... 3 BNR : Biological Nutrient Removal ............................................................................................. 31 BOD : Biochemical Oxygen Demand............................................................................................. 3 CBB : Calvin-Benson-Bassaham .................................................................................................. 16 CBOD : Carbonacious Biochemical Oxygen Demand ................................................................. 35 CHP : Combined Heat and Power .................................................................................................. 6 CNG : Compressed Natural Gas ..................................................................................................... 3 COD : Chemical Oxygen Demand ................................................................................................. ii DBD : Dielectric Barrier Discharge ................................................................................................ 2 DCW : Dry Cell Weight ............................................................................................................... 50 DME : Di methylether .................................................................................................................... 6 EDTA : Ethylenediaminetetraacetic Acid .................................................................................... 22 FaDH : Formaldehayde Dehydrogenase ....................................................................................... 17 FDH : Formate Dehydrogenase .................................................................................................... 18 GC : Gas Chromotograph ............................................................................................................. 39 GHG : Green House Gas................................................................................................................. 1 GTL : Gas to Liquid ........................................................................................................................ 8 HCCI : Homogeneous Charge Compression Ignition .................................................................... 7 IC : Ion Chromatograph ................................................................................................................ 49 ICM : Intracytoplasmic Membrane ............................................................................................... 16 LNG : Liquified Natural Gas .......................................................................................................... 3 MCFC : Molten Carbonate Fuel Cell............................................................................................ 13 MDH : Methanol Dehydrogense ................................................................................................... 21 MFC : Microbial Fuel Cell ........................................................................................................... 13 MMO : Methane Monooxygenase .................................................................................................. 2 x MSM : Mineral Salts Medium ...................................................................................................... 36 NAD : Nicotinamide Adenine Dinucleotide ................................................................................. 18 NG : Natural Gas ............................................................................................................................ 8 NOB : Nitrite Oxidising Bacteria ................................................................................................. 29 OD : Optical Density : ............................................................................................................ 37 ORC : Organic Rankine Cycle ...................................................................................................... 14 ORFC : Oxidative Reforming Fuel Cell ....................................................................................... 13 PEMFC : Proton Exchange Membrane Fuel Cell ......................................................................... 12 PHA : Poly Hydroxyalkanoates .................................................................................................... 23 PHB : Poly Hydroxybutyrate .......................................................................................................... 4 pMMO : Particulate Methane Monooxygenase ............................................................................ 16 PQQ : Pyrroloquinoline Quinone.................................................................................................. 17 RAS : Return Activated Sludge ...................................................................................................... 4 RNA : Ryboneuclic Acid .............................................................................................................. 15 RuMP : Ribulose Monophosphate ................................................................................................ 16 sMMO : Soluble Methane Monooxygenase ................................................................................. 16 SNG: Substitute Natural Gas .......................................................................................................... 8 SNR : Simultaneous Nitrification and Denitrification .................................................................. 29 SOFC : Solid Oxide Fuel Cell ...................................................................................................... 12 TRL : Technology Readiness Levels ............................................................................................ 13 TSS : Total Suspended Solid ........................................................................................................ 35 VSS : Volatile Suspended Solid ................................................................................................... 43 WAS : Waste Activated Sludge ...................................................................................................... 4 WW : Wastewater ........................................................................................................................... 4 WWTP : Wastewater Treatment Plant ............................................................................................ 1 Y : Observed Growth Yield .......................................................................................................... 41 xi CHAPTER 1: Introduction 1.1 Background Methane (CH4) is one of the major greenhouse gases (GHG) that are causing global warming directly and indirectly (Cicerone and Oremland, 1988). Methane has a life span of 12 years in the atmosphere and it has 21 to 28 times greater global warming potential than carbon dioxide (CO2) (Donner and Ramanathan, 1980). In addition, methane is responsible for 14 % of global GHG emission. Among them, 60% of global methane originates from anthropogenic sources, whereas, wastewater treatment plants (WWTPs) contribute towards 5% of the global methane emissions (Czepiel et al., 1993). Global methane emission is expected to increase by 20% over the next two decades (US EPA, 2016). WWTPs produce significant amount of methane, for instance, each year 3.9 billion tones of biomethane are produced from wastewater treatment facilities in North America (AlSayed et al., 2018c). Typically, biogas from anaerobic digesters (AD) contains 6070% methane with some impurities such as H2S and NH3. Biogas can be used as a fuel after being purified and converted into compressed natural gas or liquefied natural gas. Such processes are not cost effective as it requires high energy inputs and capital costs (Ge et al., 2014). Besides, due to the applied high pressure and methane explosive nature, it is unsafe to be stored, transferred and distributed (Ge et al., 2014). For this reason, it is necessary to develop a sustainable and costeffective technology to mitigate AD-driven biogas with sustainable value-added product recovery. The unreactive nature of methane is due to the energy of 438.8 kJ/mol required to break the C-H bond (Park and Lee, 2013). There are several thermochemical techniques with high temperature, pressure and catalyst to utilize methane gas (Park and Lee, 2013) which require intense energy use and expensive chemicals. However, this process is always involved with the generation of synthetic gas such as carbon monoxide, which is harmful to the environment. Moreover, there is 1 another process called non-thermal dielectric barrier discharge (DBD) plasma chemical process which requires high voltage and electrodes (Park and Lee, 2013). Hence, both thermochemical and plasma chemical processes are costly, less efficient and not environmentally sustainable. In contrast, biochemical process of methane utilization is a promising option as it can be operated in moderate condition with high conversion efficiency. In this process, microorganisms act as the biocatalyst which have wide biodiversity and can adapt to various environments (Ge et al., 2014). Two distinct groups of microorganisms: ammonia oxidizing bacteria (AOBs) and methane oxidizing bacteria (methanotrophs) have the ability to consume atmospheric methane gas (Hanson and Hanson, 1996). Between these groups of microorganism, methanotrophs are more advantageous due to their fast growing ability and utilizing methane as their sole carbon and energy source (Hanson and Hanson, 1996). Unlike thermochemical process, methanotrophs can activate C-H bond in methane by the methane monooxygenase (MMO) enzyme under ambient condition (Culpepper and Rosenzweig, 2012). In addition to methane mitigation, methanotrophs has attracted attention due to its wide range of biotechnological applications for value added products and processes such as methanol, biopolymers, Single-Cell Protein, Ecotine, Biodiesel, Microbial Fuel Cells, denitrification, etc (Strong et al., 2015). 1.2 Research Rationale Methane is the second most important anthropogenic source of greenhouse gas having 28 times greater global warming potential than carbon dioxide over 100 years (Stocker et al. .). Since preindustrial age, atmospheric methane concentration had increased from 0.7 ppmv to 1.7 ppmv in 1994 (El-Fadel and Massoud, 2001). Among all the anthropogenic sources WWTPs contribute about 4-5% or 20-25 Tg of CH4 year-1 to the global methane emission (Czepiel et al., 1993; El2 Fadel and Massoud, 2001). It was estimated that methane emission for municipal wastewater in typical WWTPs using activated sludge (AS) method varies from 0.039 to 0.309 kg yr−1 per inhabitant (Yver-Kwok et al., 2013). With the increase of population it is predicted that the anthropogenic methane emission will increase by 28 percent from 2005 to 2030 (HöglundIsaksson, 2012). Thus, it is important to find appropriate technology to mitigate increasing methane emissions from WWTPs. In addition, conventional AS system is criticized for intensive energy requirement where most of energy (55-57%) is required for biological treatments specially for aeration (Mamais et al., 2015; Massara et al., 2017; Robert Smith, 1978). AS process is designed to remove organic matter (BOD) by microorganism from wastewater. In this process, aerobic microorganism degrade organic waste and covert ammonia into nitrate (Ahansazan et al., 2014). For nitrogen removal additional treatment processes are incorporated which requires additional energy (Massara et al., 2017). With the increase of population, the energy demand is going to be increased over time. To keep up with the increasing energy demand it several initiatives were taken to recover energy from biogas generated from the WWTP by heat production, electricity generation by gas turbine or converting into liquid fuel like LNG or CNG. These initiatives have some limitations which are putting obstacles to methane mitigation. The major problems with biogas are: it has impurities which costs a lot to purify, it has low electricity conversion efficiency, due to its low boiling point (-164℃) it difficult to store or transport(Ge et al., 2014). To address these issues biogas can be utilized biologically by methanotrophic bacteria with some value added resource recovery (Strong et al., 2015). However, the challenge with the biological methane recovery is the integration of the system in to WWTP. 3 To incorporate methanotrophs in WWTPs several studies have been done. Methanotrophs have been successfully grown directly from the seed from return activated sludge (RAS) and waste activated sludge (WAS) (AlSayed et al., 2018a, 2018c). In addition, some study showed that methanotrophs can grow in wastewater effluent and AD centrate instead of synthetic liquid feed (Fergala et al., 2018b). Moreover, for gas feed, biogas from AD was successfully applied to methanotrophs for methanol production (Patel et al., 2016a). There is another important thing to consider in WWPs, which is the high amount of COD presence in WW. So far, no study has been done to report the behavior of methanotrophs in real WW. 1.3 Research Objective The aim of this research is to examine the growth capability of methanotrophic-heterotrophic mixed culture maintained under wastewater mainstream conditions in fed-batch mode. Specific objectives are as follows: • To enrich methanotrophic mixed culture from WWTP • To grow the enriched culture in fed-batch with ammonia and COD to reflect the main stream wastewater condition • To observe growth parameters, methane, COD and nitrogen removal continuously in repeated batch until the culture reaches to a stable condition • To investigate the nitrogen utilization pathway for determining the possibility of nitrification/ denitrification by the culture • To apply the enriched culture for methanol and polyhydroxybutyrate (PHB) production 4 1.4 Thesis Layout This thesis consists of six chapters. The first chapter is the Introduction where justification for current research including methane gas issues, methanotrophs application and their integration in WWTP, nitrogen removal issues in WWTP are provided. In the second chapter, the literature review on biogas utilization techniques, methanotrophs, methane metabolism in methanotrophs, methanol production, PHB production, nitrification-denitrification by methanotrophs are included. In the third chapter, methanotrophs growth performance and nitrogen removal activity under COD condition is provided. In the fourth chapter, two batch experiments for methanol and PHB production with COD are provided. Finally, the conclusion and future work are presented in the chapter 2 5 CHAPTER 2: Literature Review 2.1 Methane Mitigation and Utilization Techniques Wastewater and landfill are responsible for 90% of methane emissions from the waste sector which is about 18% of global anthropogenic emissions in 2004. During conventional wastewater treatment processes biogas (CH4 + CO2) is being produced through anaerobic digestion (Bogner et al., 2008). The methane containing biogas produced from anaerobic digestion is a significant source of energy (24 MJ/m3). To recover the energy in biogas several alternatives have been applied such as combustion in boilers for steam generation and heat generation for electricity and heat recovery (Monteith et al., 2005). There are various commercially feasible AD generated biogas utilization methods which include: a) electricity generation through combined heat and power (CHP) or fuel cells, b) multiple uses in industry through electricity, heat steam and cooling, c) injection into national gas grids, d) transportable fuels, e) chemical production, f) energy storage, g) cooking and lightning in rural areas, h) biohydrogen, etc. 2.1.1 Gas Engines and Turbines Biogas power plants mostly use internal combustion gas engines to produce heat and electricity. Biogas is used in other engines such as: 1) Dual fuel engines where biogas is co-fired with a small proportion of bio-diesel, bio-ethanol or bio-DME (Barik and Murugan, 2014). 2) Micro-gas turbines which require purified compressed biogas and have low combustion temperatures to produce 28% and 56% of electrical and thermal conversion efficiency, respectively (Pöschl et al., 2010). 6 3) Stirling engines are non-fuel specific external combustion engines that require less maintenance (Praetorius et al., 2009) which have 20 to 45% electricity producing efficiency. 4) Combined heat and power (CHP) is a common biogas utilizing technique in Germany. In this method produced electricity is fed in to national grid and heat is transmitted towards a district heating network. Heat also can be used for AD operation and sterilization of feedstock (Pöschl et al., 2010). 5) Homogeneous charge compression ignition (HCCI) is a process of engine operation which have alternate combustion pathway for biogas. HCCI engines has less impact of CO2 in biogas. As a result, HCCI engines have higher thermal efficiency (50%) close to diesel engine (Saxena and Bedoya, 2013). For small scale power generation, biogas fueled HCCI engine is a promising option. 6) Flameless combustion is a unique technique for combustion of different fuels. It is a mild combustion technique which is applied to alleviate unwanted emissions and improve fuel conversion efficiency (Budzianowski, 2016). Use of biogas in flameless combustion is novel but researchers found that biogas can be used effectively with this technology and it was found that electricity conversion efficiency was 53% and CHP efficiency was 82% (Hosseini and Wahid, 2013). 2.1.2 Biofuel conversion After removing the impurities in biogas, it can be converted to various biofuels which are comparable with petroleum such as biomethane, biosyngas and biohydrogen, biomethanol and bioDME (Budzianowski and Budzianowska, 2015). 7 2.1.2.1 Biomethane Biomethane is a clean bio synthetic natural gas (bio-SNG) which can be directly injected into natural gas grid for commercial use (Budzianowski, 2016). Biomethane can be obtained from biogas by separating CO2 or converting CO2 into CH4 by pressurized catalytic methanation. In Methanation of biogas CO2 is hydrogenated at 20 bar pressure with heat exchanger connected to it to remove heat produced form exothermic reaction. Hydrogen gas can be obtained through electrolysis of water with surplus renewable energy (Jürgensen et al., 2014). There are several techniques such as absorption, membrane filtration, biological method and hybrid method to remove impurities from biogas to produce clean biomethane. Moreover, Biomethane can be physically converted to various forms as liquefied natural gas (LNG), compressed natural gas (CNG) and gas to liquid (GTL). LNG is liquefied natural gas at 161℃ and 1 atm pressure. Liquefaction make 600 time reduction of gas volume which make it easy to store in an atmospheric tank and transport to the market easily (Hasan et al., 2009). In CNG, natural gas is compressed with a pressure of 1500 to 4000 psi to store it in a pressure vessel. CNG requires less volume than LNG and does not require a pipe line to transport. CNG is a simple way to utilize natural gas commercially (Young and Hanrahan, 2009). On the other hand, GTL is a technology to convert natural gas into synthetic liquid called “syncrude”. In this process NG is converted to syn-gas (CO+H2) which is then converted to a liquid mixture of hydrocarbons (syncrude) by a Fischer–Tropsch reactor with cobalt and iron as a catalyst for selling into market (LAAN and BEENACKERS, 1999). 2.1.2.2 Biosyngas and biohydrogen Biosyngas is biohydrogen rich gas which is obtained through reformation of biogas into H2 and CO. Biosyngas can be used in methanol, DME and higher hydrocarbon production. Biogas is being 8 purified for reformation as it has some impurities. For production of H2 and CO there are three principle processes: 1) steam reforming, 2) carbon dioxide (or dry) reforming, and 3) partial oxidation (Lunsford, 2000). The reformation is done in an arc gliding reactor with plasma and catalyst. In the arc gliding reactor process, biogas is converted to hydrogen enriched gas comprising 41% of hydrogen gas which have 35% thermal efficiency (Yang et al., 2009). There is an alternate method called spark-shade plasma method by Zhu et al. In reformation of biogas, the catalyst plays an important role. It was found from researches that cobalt-nickel catalyst give the best performance for biogas reformation (Xu et al., 2009). In addition, tri-reforming sorption enhanced reforming are the other options for biogas reformation (Izquierdo et al., 2013). 2.1.2.3 Biomethanol and bioDME Biomethanol is a promising sustainable fuel alternative considering its ease to storage and transport. Biogas can be converted to biomethane with the aid of catalysts but for development of suitable catalyst for direct conversion numerous researches have done. There are homogeneous, heterogeneous and enzymatic catalysts which were applied to methane gas for methanol production (Gunsalus et al., 2017). Both homogeneous and heterogenous systems have advantages and disadvantages. Homogeneous catalysts are able to break strong C-H bonds at low temperature. In this process mercury or platinum based catalysts are used with a strong oxidant like sulfuric acid or trifluoroacetic acid which are corrosive and less sustainable than O2 (Periana et al., 1998). On the other hand, heterogeneous system is based on molybdenum, iron, or copper as catalyst where O2 is used as oxidant. Molybdenum oxides is widely used with higher selection of methanol as well as stability of the product. Moreover, Iron zeolites and copper zeolites have higher methanol productivity with N2O and O2 as oxidant (Wang et al., 2017). 9 It was reported that natural gas is commercially converted to methanol by a Cu-Zn catalyst and geothermal energy (“CRI - Carbon Recycling International,” n.d.). However, conversion of biogas into biomethanol has some challenges as it has some impurities. Considering the efficiency and economy of the conversion it was suggested that biogas can be co-synthesized into biomethanol along with one or two carbon hydrocarbons. This process consists of several parallel steps like reformation, bio-Methanol synthesis, bio-DME synthesis, biohydrocarbon synthesis(optional) and fractionations (Corradini and Mccormick, 2010). There is another process for methanol synthesis with metgas, Metgas is a mixture of H2 and CO with a 2:1 ratio which is used in methanol synthesis with NiO/MgO as catalyst at 5-30 atm pressure and 800-950℃ temperature (Olah et al., 2013). 2.1.2.4 Ethylene Methane can be converted to ethylene by oxidative coupling of methane (Holmen, 2009). In this process methane is coupled with O2 to form ethylene with water, ethane and other higher order hydrocarbons. The reaction is catalyzed by different heterogeneous catalysts based on Li/MgO, Fe2O3, and La2O3 at 700℃ temperature. This process generates a significant amount of ethane, C3 products, CO, and CO2 along with ethylene. There is an alternative way to produce ethylene by heating methane in absence of O2 which generate ethane and H2 but no oxygenates are formed (Holmen, 2009). Ethane can be upgraded to higher value ethylene by oxidative dehydrogenation where higher temperature >500℃ and tantalum hydride catalyst supported on SiO2 is applied. In this process along with ethylene other higher hydrocarbons can be produces as by-product. The problem of this process is formation of hydrogen-deficient CHx fragments and selection of catalyst with appropriate temperature. 10 2.1.2.5 Aromatics/ Higher hydrocarbon Benzene is a precursor of range of aromatic compounds. Usually benzene is derived from petroleum-based compounds. However, there is a process called methane dehydroaromatization which was discovered in 1993 (Wang et al., 1993). In this process methane is converted into benzene and H2 gas with no oxygenate including CO and CO2. As a result, H2 can be used directly in fuel cells. For the reaction, 900℃ temperature and zeolite-based catalyst such as Mo-ZSM-5 is used. The catalyst activates carbon bond and helps to form aromatic compounds. As the thermodynamically favorable condition of benzene is similar to naphthalene, naphthalene is formed as by-product in this process (Tang et al., 2014). The disadvantage of this process is that it requires high temperature to make the reaction happen. In addition, significant amount of coke is formed which makes the catalyst to be regenerated with H2. Further research is required to apply this method efficiently. There is another process to synthesize higher hydrocarbons from biogas using Al-Co-Ni-Cr based catalysts (Gunnerman and Gunnerman, 2009). In this process biogas is fed into the first reactor where the catalyst is immersed into a liquid phase. The product gas is vaporised and drawn from the reactor and condensed and then fed into a second reactor. The condensate liquid is separated, and non-condensable gases are recycled back to first reactor. In this process the main synthesized products are paraffins (25-35%), naphthenes (58-63%) and aromatics (6-17%). The major issue with this process is the energy efficiency needed to consider while upscaling the system. 2.1.3 Fuel Cells Fuel cells are innovative technologies for converting chemical energy of fuel into electricity with higher efficiency and less harmful emissions (Perry Murray et al., 1999). Biogas can be applied 11 in fuel cells as fuel but since biogas has impurities, it is required to condition it before using it in fuel cells. There are various fuel cells where biogas can be applied for power generation. 2.1.3.1 SOFCs Solid oxide fuel cell (SOFC) produce electricity by oxidizing fuel where solid oxide or ceramic construction materials are used. This process requires high operating temperature and long starting time which make it less operationally flexible. However, this process is resistant to biogas fuel impurities (Shiratori et al., 2008). It was found that at 1000℃ SOFC can tolerate biogas with up to 1 ppm H2S. The problem of SOFC is excessive deposition of carbon in to the reactor. It was claimed that biogas diluted with air fed in to SOFC can prevent carbon deposition without compromising cell voltage (Shiratori et al., 2010). Moreover, in a study three differently conditioned biogas systems were applied to SOFC in order to prevent carbon deposition (Farhad et al., 2010). It was found from the study that partial oxidation was the best approach with 80.5% CHP conversion efficiency. In another study it was suggested that prior to SOFC application, biogas can be enriched with hydrogen gas which can lower carbon deposition (Lanzini and Leone, 2010). This study also claimed that at certain threshold of hydrogen level carbon deposition stops and for sustainable power generation hydrogen can be obtained through electrolysis of water. 2.1.3.2 PEMFCs Unlike SOFC, proton exchange membrane fuel cell (PEMFC) requires low temperature (50100℃) and uses a special polymer electrolyte membrane. Biogas is less suitable for PEMFC as it requires clean fuel. There are several techniques developed by scientists to utilize biogas into PEMFCs. In an experiment, biogas was reformed under steam to produce hydrogen with 50% 12 purity and the reformed gas was efficiently applied to PEMFC with stable performance (Schmersahl et al., 2007, p.). In another study high temperature PEMFC (HT-PEMFC) with a modular stack was suggested (Birth et al., 2014). In this process, biogas is reformed to yield hydrogen gas for downstream oxidation. The electrical efficiency of HT-PEMFC system was found to be 40%. There is another process called oxidative reforming fuel cell (ORFC) system where biogas is converted to a mixture of CO2 and H2 (Budzianowski, 2010). The gas mixture is separated to form hydrogen enriched gas which is finally sent to PEMFC for electricity generation. The calculated electrical efficiency is 56% which is higher than gas turbine cycles. The problem of this system is that it has low Technology Readiness Levels (TRL) to commercialize. 2.1.3.3 MCFCs Molten carbonate fuel cell (MCFC) is a high-temperature fuel cell that uses electrolyte of molten carbonate salt mixture suspended in a porous ceramic matrix. MCFC operates at higher temperature (600-700℃) which enables use of cheaper catalysts based on Ni (Milewski et al., 2014). Biogas can be used in MCFC as it requires CO as fuel for this type of cells. However, H2S required to be filtered for MCFC use. A combined heat, hydrogen and power (CHHP) was developed with MCFC system for sustainable biogas utilization (Hamad et al., 2014). In this system electricity can be use for local use, heat for biogas digester and hydrogen can be transported to the market. 2.1.3.4 MFCs Microbial fuel cell (MFC) is technology to produce direct electricity or energy carriers from various organic substrate. In a study MFC was used to treat primary digested sludge where both biogas and electricity production were observed (Ge et al., 2013). Moreover, it was reported in several studies that methane gas can be used as an energy source to drive MFC for direct electricity 13 production (Chen et al., 2018). However, the MFC system needs further research to minimize capital cost and stable performance in order to scale up for industrial use. 2.1.3.5 ORCs Organic Rankine Cycle (ORC) is technique to utilize organic energy in liquid phase by lowering boiling point usually less than water steam. Using ORC the best energy conversion from biogas was achieved at 1.5 to 2 compression ratio with air temperature 335 to 340K (Di Maria et al., 2014). Biogas to electricity conversion efficiency was 20% which was lower than gas turbine efficiency. In order to utilize biogas for ORC it is required more investment and further research be conducted to increase energy conversion efficiency. 2.1.3.6 Hybrid energy system Hybrid energy system is a combination of two or more energy systems to maximize fuel to energy conversion efficiency. For biogas SOFC-GT hybrid system was suggested in the literature. In this system SOFC provides high fuel to electricity conversion ratio and GT ensures maximum fuel utilization and exploits thermal energy derived from high temperature by SOFC. By optimizing biogas fed SOFC-GT hybrid system 65% of biogas to energy conversion was achieved (Wongchanapai et al., 2013). Moreover, it was feasible to produce 55% electrical efficiency and more than 80% CHP efficiency. The problem of hybrid systems are that they are expensive and not feasible for small scale biogas plant. 2.1.4 Biological methane mitigation Compared to physical and chemical processes, biological methane utilization processes require less energy inputs. Various methanotrophic bacteria can utilize methane as their sole carbon and energy source. Methanotrophs can oxidize methane under both aerobic and anaerobic conditions. 14 Aerobic methanotrophs use oxygen as electron donor to oxidize methane terminally into CO2 with methanol, formaldehyde and formate as intermediates (Hanson and Hanson, 1996). Based on the metabolic pathways, methanotrophs have various engineering application with methane utilization. In the next section background of methanotrophs, methane utilization mechanism and various applications will be discussed. 2.2 Methanotrophs Methanotrophs are the organisms that can utilize single carbon source organic compound (C1) such as methane and methanol (Brock Biology of Microorganisms, 14th Edition, n.d.). Methanotrophs were first discovered in 1906 by Söhngen and later it was successfully isolated and classified in 1970 (Whittenbury et al., 1970). Methanotrophs utilize methane both` aerobically and anaerobically as a sole carbon and energy source. They are gram-negative bacteria that belong to Proteobacteria under the group of prokaryotes (Hanson and Hanson, 1996). Methanotrophs naturally exist in diverse environments such as: soils, peat bogs, wetlands, sediments, lakes, waste waters, fresh waters, and marine waters (Hanson and Hanson, 1996). Generally aerobic methanotrophs oxidizes methane in to carbon dioxide through a series of intermediates including methanol (CH3OH), formaldehyde and formate (Hanson and Hanson, 1996). 2.2.1 Methanotrophs classification Methanotrophs can be classified as aerobic and anaerobic methanotrophs depending on their terminal electron acceptor. In addition, based on the carbon assimilation pathways, cell morphology, membrane arrangement, 16S RNA sequences, and other metabolic characteristics methanotrophs are classified as Type I, Type II and Type III (Hanson and Hanson, 1996; Knief, 2015). 15 Type I methanotrophs are Gamma subdivision of Proteobacteria which have Methylococcaceae and Methylothermaceae families. Type Imethanotrophs can be found in various environment such as soils, landfills, sewage and activated sludge, denitrification reactors, anaerobic digesters (Bowman, 2014; Ho et al., 2013). Type I methanotrophs have well developed intracytoplasmic membrane (ICM) which enables them to produce mostly particulate methane monooxygenase (pMMO) (Bowman, 2014). For carbon assimilation they use formaldehyde (CHOH) trough ribulose monophosphate (RuMP) cycle (Bowman, 2006). Moreover type I methanotrophs have high growth rate and have higher methane oxidation efficiency (Fergala et al., 2018a). Methylobacter, Methylomicrobium, Methylocaldum, Methylococcus, Methyloglobulus, Methylomonas, Methylosphaera, etc. are some strains of type I methanotrophs (Knief, 2015). On the other hand, type II methanotrophs are Alpha subdivision of Proteobacteria phylum can be found in soil, freshwater sediments, landfills, sewage sludge (Bowman, 2006; Ho et al., 2013). Type II methanotrophs have the ICM aligned to the cell periphery which have the ability to express soluble methane monooxygenase (sMMO) (Semrau et al., 2010). For cell synthesis they assimilate carbon from formate(CHOOH) through Serine cycle (Bowman, 2006). Unlike type I methanotrophs they are slow to grow and have the unique ability to accumulate biopolymer under nutrient limited condition (Bowman, 2006; Henckel et al., 2000). Methylosinus, Methylocystis are the strains of type II methanotrophs. Type III methanotrophs are Verrucomicrobia can be found in hot acidic geothermal environment (Knief, 2015). Verrucomicrobia do not have ICM system but they possess pMMO with unidentified location inside their cell (van Teeseling et al., 2014). They assimilate carbon from Carbon dioxide via Calvin-Benson-Bassaham 16 (CBB) cycle. Methylacidiphilum and Methylacidimicrobium genus are the examples of type III methanotrophs . (AlSayed et al., 2018d; Knief, 2015). 2.2.2 Methane assimilation by methanotrophs In methane oxidation by methanotrophs, methane is terminally oxidized in to carbon dioxide through series of intermediates with the aid of various enzyme secreted from the bacteria (Hanson and Hanson, 1996). Initially, methane is oxidized in to methanol by MMO enzyme which reduces the oxygen molecule into two monovalent oxygen atoms. One oxygen atom breaks the C-H bond in methane forming methanol. While the other oxygen atom is converted to water. MMO enzyme has two forms: sMMO and pMMO. sMMO uses nicotinamide adenine dinucleotide NAD(P)H as electron donor generated from formaldehyde and formate oxidation (Hanson and Hanson, 1996). The electron donor for pMMO is not clear but is assumed that pMMO has various electron source such as ubiquinol (Q8H2) (Kalyuzhnaya et al., 2015). sMMO expressing cells have higher efficiency in methane oxidation and pMMO containing cells have broad substrate range (Chistoserdova et al., 2005; Kalyuzhnaya et al., 2015). 𝐶𝐻4 + O2 + 2e − + 2H+ → 𝐶𝐻3 OH + 𝐻2 O Methanol is further oxidized into formaldehyde through quinoprotein methnol dehydrogenase (MDH) (Chistoserdova et al., 2005). During this reaction pyrroloquinoline quinone (PQQ) is reduce to PQQH2 which later act as a electron donor for pMMO or other electron acceptor (Hanson and Hanson, 1996). Formaldehyde is an important intermediate in methanotrophs metabolism as part of it is terminally oxidized into carbon dioxide and part of it is assimilated for cell synthesis through RuMP or Serine pathway (Hanson and Hanson, 1996). Formaldehyde is oxidized into formate by formaldehayde dehydrogenase (FaDH) which is either nicotinamide adenine 17 dinucleotide (NAD)-linked or PQQ containing cytochrome-linked enzyme. Formate is finally oxidized into carbon dioxide which is catalyzed by NAD dependant enzyme formate dehydrogenase (FDH) (Smith et al., 2010). Methanol is further oxidized in to formaldehyde. Subsequently, part of formaldehyde is assimilated for new cell production and part of formaldehyde is further oxidized into formate by NAD (P) linked aldehyde dehydrogenase. Finally, part of formate can be assimilated for cell synthesis, whereas the remaining part is oxidized into CO2 (Hanson and Hanson, 1996; Hwang et al., 2018). 2.2.3 Factors affecting methanotrophs growth The growth rate varies for different types of methanotrophs. Type I methanotrophs are the fastest and type III are the slowest to grow (van Teeseling et al., 2014). Several factors affect the growth of methanotrophs such as: pH, temperature, substrates, methane to oxygen ratio, methane solubility, nitrogen source, copper, etc. 2.2.4 pH Most of the methanotrophs grows in the pH range of 5.5 to 8 but there are some genus like verrucomicrobial methanotrophs that can grow in pH 1.5 to 3.5 and methylomicrobim species can grow in pH 8 to 10 (Bowman, 2006; van Teeseling et al., 2014). Methanotroph do no require sodium chloride for growth but they can tolerate up to 7 mg/L of NaCl (van der Ha et al., 2010). 2.2.5 Temperature Methanotrophs mostly grow in the temperature rage of 20 to 30℃ however, most type I and type III methanotrophs prefers higher temperature. For example type I methylococcus genus can grow 18 optimally between 42 to 55 ℃ (Bowman, 2006). On the other hand, all type II methanotrophs can survive at lower temperature range 4 to 10 ℃ (Bowman, 2006). 2.2.6 Substrate Methanotrophs usually take methane as their substrate but they can utilize other C1 compounds such as methanol, formate and methylamines as substrate (Bowman, 2014; Hanson and Hanson, 1996). Interestingly it was discovered that some type II methanotrophs like methylocella species can consume multiple carbon source such as acetate, ethanol, malate, succinate, etc. Moreover, methylocella silverstris can grow faster on acetate than methane (Semrau et al., 2011). 2.2.7 Methane to oxygen ratio Methane to oxygen ratio is not a decisive parameter for growing specific type of methanotrophs. Type II methanotrophs can form stable slow growing community at above 1% methane concentration (Semrau et al., 2010). In addition, they can also dominate at methane concentration less than 0.06%. However, some studies reported that type II methanotrophs prefers to grow in high methane to low oxygen condition. On the other hand, type I methanotrophs usually dominates in the enrichment process as they are faster growing than type II (Semrau et al., 2010). So, both type of methanotrophs has the ability to grow in different methane and oxygen concentration. 2.2.8 Nitrogen source All type II methanotrophs and some type I methanotrophs can fix atmospheric nitrogen via oxygen sensitive nitrogenase (Bowman, 2006). In addition, type II methanotrophs dominate in N-limited condition whereas type I methanotrophs prefer higher nitrogen condition. Mostly, methanotrophs like to grow on inorganic nitrogen, nitrate, ammonia, etc. Methanotrophs can tolerate up to certain concentration of ammonia as high ammonia content can inhibit the growth of methanotrophs. 19 Ammonia competes for MMO enzyme and produce toxic hydroxylamine or nitrite. In contrast, nitrate support higher growth rate for both type of methanotrophs. Some study show that ammonia has less toxic effect on type II methanotrophs which help them to form stable community. So, nitrate can be selected as nitrogen source for enrichment of type I methanotrophs and ammonia or nitrogen limited condition can be selected for type II methanotrophs. 2.2.9 Copper Copper can control the form of MMO enzyme in pMMO or sMMO depending on the concentration level of copper (Semrau et al., 2010). However, copper is not a decisive parameter to enrich specific type of methanotrophs as both type I and type II can express pMMO enzyme (Cantera et al., 2016). Some studies showed that with the addition of copper had significantly increased the methane uptake rate for methanotrophic mixed culture (van der Ha et al., 2010). However high copper concentration can inhibit the growth of methanotrophs due to its toxicity. 2.3 Application of methanotrophs in WWTPs Methanotrophs are promising, advantageous microorganism that they can be applied to produce product such as bio fuel, bio polymer, etc. They can be applied in certain bio process such as methane mitigation, contaminant bioremediation, denitrification, electricity generation in microbial fuel cells, etc (Strong et al., 2015). Methanotrophs have many aspects in WWTPs among them methanol, biopolymer production and nitrification/denitrification by methanotrophs will be discussed. 20 2.3.1 Methanol production by methanotrophs Due to storage, transportation difficulties and low electricity conversion efficiency (25-45%), methane is not a good option for direct use a fuel (Bachmann, n.d.). In contrast, methanol has higher transportability, security, high energy content with higher efficiency making it a lucrative option as fuel. In addition, methanol is used as external carbon source for denitrification process in WWTPs (Strong et al., 2015). So, methanol can be considered as sustainable alternative option for methane. Methanotrophs oxidizes methane into methanol which is further oxidized consecutively to form carbon dioxide with formaldehyde and formate as intermediates. Methanol oxidation is catalyzed by methanol dehydrogense (MDH), formaldehyde dehydrogenase (FaDH) and formate dehydrogenase (FDH) subsequently (Sheets et al., 2017). To get methanol, the oxidation of methanol to formaldehyde is prevented by inhibiting the activity of (methanol dehydrogenase) MDH enzyme by sodium chloride, phosphate, etc (Hwang et al., 2014). Inhibition of MDH activity results in a shortage of electrons which is required for cellular energy and continuous methane oxidation. As a result, external electron source (formate) is added to complete the reaction. The reported methanol production from pure culture is 0.6-1120 mg/L with conversion efficiency 27-80% (Ge et al., 2014). In addition, methanol production from mixed culture achieved was 240~485 mg/L (AlSayed et al., 2018d). Factors affecting methanol production: Methanol production is affected by several factors such as: head space gaseous composition, MDH inhibitors and biomass density (Patel et al., 2016b). It is important to know how these factors affect methanol productivity to scale up the methanol production in a industrial scale. 21 Nutrients Copper is an important nutrient for methanol production as copper support the expression of pMMO enzyme. With the addition of 5 µM of copper ion significantly increases methanol production but more than 10 µM inhibits the growth and methanol production. In addition, it is reported that addition of iron also increases the methanol productivity (Sheets et al., 2016). Gas mixing ratio Theoretically to produce one mole methanol it requires one mole methane and one mole oxygen gas. However, for longer incubation period more methane gas increases methanol production. Moreover, with increased methane concentration methane uptake rate increases. MDH inhibition Methanol in methanotrophs is oxidized by PQQ linked MDH enzyme (Hanson and Hanson, 1996). MDH inhibition is crucial part to prevent methanol oxidation. For MDH inhibition several chemicals such as Phosphate, NaCl, Cyclopropanol, EDTA, MgCl2 and NH4Cl are used separately or in combination (Sheets et al., 2016; Yoo et al., 2015). The problem with MDH inhibitors is they inhibit MDH activity and MMO activity as well (Ge et al., 2014). As a result, methanotrophs growth and methane uptake is negatively affected by MDH inhibitors. So, it is very important to choose suitable inhibitor to maximize methanol production. Phosphate is commonly used inhibitor and can be used with other inhibitor such as NaCl, EDTA, MgCl2. It was found that with increased concentration of phosphate along with MgCl2 increases methanol production as mgCl2 support sMMO activity and cellular growth (Duan et al., 2011). In addition, NH4Cl and NaCl have high methane to methanol conversion efficiency but they reduce the MMO activity (Yoo et al., 2015). 22 External electron donor In methane oxidation, to produce one mole methanol 2 moles of electrons are required which usually come from afterward oxidation of methanol. Due to application of MDH inhibitor external electron source is required. Formate and formaldehyde can used as electron donor, but formaldehyde has toxic effect on methanotrophs. So, formate is a suitable source for external electrons(Hwang et al., 2015). The problem with formaldehyde is the cost to apply it in a industrial scale. Biomass density With the increase of biomass density from low to high increase methanol production to a certain limit, after that increase in cell density negatively affect methanol production (Lee et al., 2004). However, some study showed that with increased MDH inhibitor methanol production was increased at higher concentration of biomass. In addition, increasing methane concentration with increased biomass density also resulted higher methanol production (Lee et al., 2004). Biopolymer production by methanotrophs Bioplastic or biopolymer is a green alternative to conventional plastic products as biopolymer is produced naturally and degradable after some time. Most of the bioplastics belongs to the family of Polyhydroxyalkanoates (PHA) where polyhydroxybutyrate (PHB) is a member among them. PHA accumulating methanotrophs can accumulate PHB using methane gas under nutrient limiting condition (Strong et al., 2015). Under aerobic condition, methanotrophs without essential nutrients such as nitrogen, phosphorus are forced to store PHB inside their cell for surviving (Karthikeyan et al., 2015). 23 2.3.2 PHB production mechanism in methanotrophs Figure 2.1 PHB production pathway in Methanotrophs. (Adapted from AlSayed et al., 2018b) In PHB production acetyl-CoA is the main intermediate which is produced through serine pathway (Babel, 1992). This means PHB production might be exclusive to type II methanotrophs. Normally, in balanced condition with all nutrient, acetyl-CoA goes towards TCA cycle to fulfill energy needs. While in nutrients limiting condition, the bacteria adopt survival strategy by switching towards PHB cycle for cell maintenance. In PHB cycle, at first, acetyl-CoA is converted to Acetoacetyl-CoA which is reduced to β-hydroxybutryl-CoA with the help of β-ketothiolase and Acetoacetyl-CoA reductase enzyme. β-hydroxybutryl-CoA is then converted by a polymerase, PHB synthetase, to form PHB. PHB granules is depolymerized to Hydroxybutrate monomers by PHB depolymerize enzyme. After this step, β-hydroxybutyrate dehydrogenase enzyme converts Hydroxybutrate monemers into acetoacetate which is finally converted in to Acetoacetyl-CoA by Acetoacetate succinyl-CoA transferase enzyme to complete the whole cycle (Karthikeyan et al., 2015). 24 Factors affecting PHB production: PHB accumulation is mainly done by type II methanotrophs. For PHB production, growth parameters should be selected to favor the growth of type II methanotrophs. Here the factors for type II selection and PHB accumulation will be discussed. Nitrogen source It has been proven through different studies that nitrogen limited condition is a reliable parameter for long term PHB productivity. Methanotrophs can grow on inorganic nitrogen such as ammonia, nitrate and nitrogen as their nitrogen source. However, as discussed earlier, for PHB accumulation selection of type II methanotrophs is an important factor. From studies it was found that strains of methanotrophs methylocystis parvus OBBP and methylocystis GB25 (in mixed culture) grown on ammonia can produce more PHB than grown on nitrate (Rostkowski et al., 2013; Wendlandt et al., 2001). Previous studies also suggest that methanotrophs grown on nitrate have higher biomass density but produce less PHB due to invasion of type I methanotrophs. On the other hand, higher PHB production was observed with ammonia but less biomass density results. Considering these factors, a strategy was developed to grow type II methanotrophic mixed culture from activate sludge with ammonia as nitrogen source then increase the biomass density using nitrate as nitrogen source. This technique yielded about 40% nitrogen which could be increased through optimization of nitrogen concentration (Criddle and Sundstrom, 2015). Phosphorus Phosphorus is important for Type II methanotrophs selection. Phosphorus concentration up to 225 mmol helps to maintain sMMO activity which is expressed through type II methanotrophs. However, for PHB accumulation phosphorus limited condition is required. It was observed that in 25 phosphorus limited condition 46% PHB accumulated by methylocystis GB25 starin(Wendlandt et al., 2001). Copper The effect of copper on PHB accumulation has contradictory studies. Some study showed that reducing copper from 15 to 5µM increased PHB accumulation from 18% to 49% (Sundstrom and Criddle, 2015) . However, in another study it was found that addition of 5µM copper increased PHB accumulation from 25% at without copper to 51% (Zhang et al., 2017). Other nutrients It was found that iron concentration from 40 to 80 µM have positive effect on sMMO activity (Park et al., 1991). In addition, sodium, potassium, magnesium and mercury have inhibitory effect on PHB production (Wendlandt et al., 2005). Temperature & pH In some studies it was found that temperature more than 30℃ reduce the activity of sMMO and more than 45℃ reduce the activity of pMMO (Park et al., 1991). pH in the range of 6 to 7 is suitable for methanotrophs growth however, pH 5 can be used to promote the growth of type II methanotrophs (Pieja et al., 2011). Methane and oxygen At high level of oxygen cause increase in oxidation of methanol to formaldehyde produce inhibitory effect on metabolic activity of methanotrophs (Costa et al., 2001). In addition, with low methane concentration where ammonia is a nitrogen source increase hydroxylamine production 26 causing both ammonia and hydroxylamine toxicity on biomass. In toxic condition bacteria are forcing to store PHB inside their cell to survive under harsh condition. 2.3.3 Nitrification and denitrification by methanotrophs It has been discovered that several strains of methanotrophs can nitrify and denitrify using nitrogen compounds. In this section the mechanism for nitrification and denitrification and the enzymes involved will be discussed. 2.3.3.1 Nitrification by methanotrophs using ammonia as nitrogen source MMO enzyme in methanotrophs have similar properties as Ammonia monooxygenase (AMO) enzyme that come from ammonia oxidizing bacteria (AOB). Due to this unique ability methanotrophs are able to oxidize ammonia through MMO enzyme. In this process ammonia competes with methane for MMO enzyme for oxidation (He et al., 2017). In the first step ammonia is oxidised into hydroxylamine intermediate by MMO enzyme. Hydroxylamine is highly toxic for methanotrophs which make the methanotrophs to take detoxification strategies. One strategy is to oxidize hydroxylamine in to nitrite by hydroxylamine oxidoreductase (HAO) enzyme and, another strategy is to reduction of hydroxylamine back to ammonia by hydroxylamine reductase (Stein and Klotz, 2011). 27 a→ methanemono-oxygenase; b → HAO and unknown enzymes; c → NorB nitric oxide reductase and unknown enzymes; d → NirK, NirS and HaoA’ nitrite reductases and unknown enzymes; e → hydroxylamine reductase; f → NirB assimilatory nitrite reductase → Figure 2.2: nitrogen catabolic pathways in aerobic methanotrophic bacteria Nitrite is also toxic for which methanotrophs take detoxification strategy by reducing it to nitric oxide by NirK, NirS and HaoA nitrite reductases and unknown enzymes or reduce back to ammonia by NirB assimilatory nitrite reductase (Stein and Klotz, 2011). Some methanotrophs were found to resist the toxicity of ammonia, hydroxylamine and nitrite. It was found that some strains M. album from type I and Methylocystis sp. And M. sporium from type II were able to produce nitrite from ammonia (Nyerges and Stein, 2009). In another study 14 genotypically different methanotrophs strains were tested to study their nitrogen fixation metabolism and tolerance against ammonia, hydroxylamine and nitrite. It was found that most of the strains can tolerate up to 40 mM of NH4Cl, 2 mM of NaNO2 and 1 mM hydroxylamine. In addition, all type I strains produced N2O from hydroxylamine oxidation and all type II strains produced N2O from nitrite oxidation (Hoefman et al., 2014). 2.3.3.2 Denitrification by methanotrophs Methanotrophic can fix nitrogen through denitrification directly or indirectly. Aerobic methane oxidation coupled to denitrification (AME-D) is a widely discussed process where methanotrophs indirectly helps denitrification. In AME-D methanotrophs oxidize methane aerobically and produce organic compounds such as methanol, acetate, citrate and proteins which are utilized by the denitrifiers in WWTP as electron donor for denitrification (Modin et al., 2007). During this 28 process the oxygen level is kept at a minimum to keep aerobic methanotrophs active along with no inhibitory effect on the denitrifiers. There is another indirect process called simultaneous nitrification and denitrification process (SNR) where methanotrophs, autotrophic nitrifiers and dentrifiers live together in a same bioreactor under same operating condition (Lee et al., 2001). Recently it has been discovered that methanotrophs can denitrify by themselves under anoxic condition. Denitrification in methanotrophs can be explained as energy conservation strategy for respiration during oxygen limited condition and switching their electron acceptor from oxygen to nitrate or nitrite (Kits et al., 2015). Denitrification in methanotrophs is mainly partial denitrification because most of the strains tested were able to produce nitrous oxide terminally. For example, Methylomonas denitrificans FJG1 can do partial denitrification along with methane oxidation while producing nitrous oxide as terminal gas (Zhu et al., 2017). However, only one strain was found that was able to perform complete denitrification. Mythalocystis sp. SC2 strain was found to have nitrous oxide reductase (NOR) to convert nitrous oxide into nitrogen gas (Dam et al., 2013). 2.3.4 Nitrogen removal practice in WWTPs Two approaches are followed in WWTPs for nitrogen removal: conventional nitrificationdenitrification process and shortcut nitrogen removal process. Nitrification-denitrification process is most common in conventional WWTPs. Nitrification is a two-step process where ammonium is oxidised first to nitrite (NO2−) by ammonium oxidising bacteria (AOB) and then to nitrate (NO3−) by nitrite oxidising bacteria (NOB) under aerobic condition (Daims et al., 2016). In denitrification, under oxygen limited condition, denitrifying organisms reduce the nitrate terminally into nitrogen gas (Kraft et al., 2011). Aeration is needed for nitrifying bacteria what makes it energy intensive process. Whereas, the denitrifiers use organic substances such as methanol as a carbon source 29 which requires the addition of external carbon (Zhu et al., 2017). On the other hand, the process of shortcut nitrogen removal was adopted which minimize or eliminate aeration and external carbon addition in the treatment process. There are several shortcut nitrogen removal processes such as: Nitritation-denitritation process, Deammonification and ammonia oxidation process by anaerobic ammonia oxidizing bacteria (Anammox). In nitritation-denitritation process nitrite oxidation to nitrate is bypassed to lower the oxygen requirement in the bioreactor. The limitation of this process is to inhibit the activity of NOBs to prevent further oxidation of nitrite into nitrate (Al-Omari et al., 2015). In Deammonification ammonium is oxidised directly to nitrogen gas by annamox bacteria without the need for carbon and in the absence of oxygen. The challenge for deammonification is that annamox is slow growing and it requires nitrite as oxidant which come through partial nitrification of ammonia by AOBs. Deammonification is more suitable to side stream treatment where ammonium concentration is high (Fernández et al., 2016). Collectively, both conventional and shortcut nitrogen removal processes expose different challenges that hindering it from being adopted in WWTP. Those challenges include the addition of external carbon source, extra energy input, controlling bacterial activity while nitritition, nitrogen removal in high COD environment. 2.3.5 Integration of methanotrophs in WWTPs The best way to integrate methanotrophs in WWTP is to grow them within the system. WWTPs provide most of the elements required for methanotrophs cultivation. First of all, the seed required for methanotrophs cultivation can be obtained from waste activated sludge (WAS) or return activated sludge (RAS) during typical wastewater treatment processes. Secondly, for liquid feed WW effluent containing low level N, C and P can be used instead of synthetic feed. In addition, AD centrate contains high concentrated nutrients which can be used as feed by dilution. 30 Figure 2.3: Integration of Methanotrophs in to WWTP Moreover, methanotrophs can be grown with mainstream WW influent as feed containing COD, ammonia and phosphorus. Thirdly, the biogas generated from the anaerobic digester (AD) can be used as feed for methanotrophs growth and also can be used as external carbon source for denitrification process in Biological Nutrient Removal (BNR) system. Using the biogas methanotrophs will grow in the reactor and produce organic compounds to be used by the denitrifiers. Fourthly, Methanotrophs that are cultivated from the process can be utilized for methanol production or PHB production. PHB enriched methanotrophs can be utilized either in a BNR reactor for denitrification or can be used as external electron source instead of costly formate in methanol production. So, integration of methanotrophs is beneficial as it can produce valuable products such as PHB and Methanol, and it can assist nitrification and denitrification directly or indirectly. These modifications can make onto WWTPs energy self-sufficient by saving and producing fuel. In 31 addition, as this process will increase nitrogen removal efficiency from main stream wastewater which will make the process more sustainable, environment friendly and safe to public health. 32 CHAPTER 3: Methanotrophic-heterotrophic mixed culture performance under varying chemical oxygen demand (COD) 3.1 Introduction Global methane emission is expected to increase by 20% over the next two decades (US EPA, 2016). WTPs produce significant amount of methane, for instance, each year 3.9 billion tones of biomethane are produced from wastewater treatment facilities in North America (AlSayed et al., 2018c). Typically, biogas from anaerobic digesters (AD) contains 60-70% methane with some impurities such as H2S and NH3. Biogas can be used as a fuel after being purified and converted into compressed natural gas or liquefied natural gas. Such processes are not cost effective as it requires high energy inputs and capital costs (Ge et al., 2014). Besides, due to the applied high pressure and methane explosive nature, it is unsafe to be stored, transferred and distributed (Ge et al., 2014). For this reason, it is necessary to develop a sustainable and cost-effective technology to convert AD-driven biogas into more sustainable value-added product. There are several thermochemical techniques with high temperature, pressure and catalyst to utilize methane gas (Park and Lee, 2013) which require intense energy use and expensive chemicals. However, this process always involved with generation of synthetic gas such as carbon monoxide which is harmful to the environment. Moreover, there is another process called nonthermal dielectric barrier discharge (DBD) plasma chemical process which requires high voltage and electrodes (Park and Lee, 2013). Hence, both thermochemical and plasma chemical process is costly, less efficient and not environmentally sustainable. 33 3.2 Biochemical conversion of methane by methanotrophs In contrast to physical and chemical processes, biochemical process of methane utilization is a promising option as it can be operated in moderate condition with high conversion efficiency. In this process, microorganisms act as the biocatalyst which have wide biodiversity and can adapt to various environment (Ge et al., 2014). Methanotrophs can grow on methane as their sole carbon and energy in which C-H bond with is activated by the methane monooxygenase (MMO) enzyme under ambient condition (Culpepper and Rosenzweig, 2012). 3.3 Nitrogen issues in WWTPs Widely used conventional Activated Sludge (AS) system is criticized for intensive energy requirement where most of energy (55%) is required for biological treatment only (Mamais et al., 2015; Massara et al., 2017). AS process is designed to remove organic matter (BOD) by microorganism from wastewater. In this process in presence of oxygen microorganism utilize organic waste and also covert ammonia into nitrate (Ahansazan et al., 2014). For nitrogen removal additional treatment processes are incorporated which required additional energy (Massara et al., 2017). Ammonia (NH3-N) is the major form of nitrogen that present in typical wastewater. Typical municipal wastewater influent contains 20~60 mg/l total N including 60% NH3-N and 300~500 mg/l COD (Chai et al., 2015; Gilbert et al., 2014; Hanson and Lee, 1971). Considering domestic wastewater dominance most of the treatment plant around the world tend to treat it biologically by activated sludge method (Krishna Reddy et al., 2017). The total nitrogen removal rate is around 50% during the biological treatment in conventional activated sludge process through cellular assimilation (Liu et al., 2018). As a result, treated wastewater effluent may contain up to 15~30 34 mg-N/L directly in to water bodies which are mainly in the form of nitrate (Tchobanoglous et al., 1991). WTPs in GTA area mainly remove CBOD, Phosphorus, TSS and E. Coli (City of Toronto, 2017). Nitrogen is considered as one of the major contributors to poor water quality and its (Purwono et al., 2017). Nitrogen presence provide the needed nutrients the growth of algae causing algal blooms, which depletes the dissolved oxygen in the water bodies needed for the aquatic. Such phenomenon is called eutrophication. Moreover, nitrogen presence in drinking water in excessive concentrations may cause serious public health issues (Obaja et al., 2003; Ward et al., 2018). 3.4 Potential application of Methanotrophs in WWTPs In WWTP methanol can be produced from biogas generated from anaerobic diester. In biogas the presence of carbon dioxide can help MDH inhibition. The reported maximum methanol production from mixed culture achieved was 240~485 mg/L (AlSayed et al., 2018d). In addition, Methanotrophs without essential nutrients such as nitrogen, phosphorus can convert methane into PHB and store them inside their cell (Karthikeyan et al., 2015). PHB is extracted from the bacterial cell processed to produce polymer to be used in industries. The advantage of PHB is that it is biodegradable after certain period of use (Rostkowski et al., 2012). The reported PHB accumulation percentage ranges between 40 to 60% of their cellular weight (Fergala et al., 2018b; Khosravi-Darani et al., 2013). PHB can be used as electron donor in methanol production. Moreover, produced methanol can aid denitrification activity by providing carbon to the denitrifiers. From recent discovery, methanotrophic bacteria can do nitrification and partial denitrification along with methane oxidation while producing nitrous oxide as terminal gas (Zhu et al., 2017). 35 PHB is an eco-friendly bio-polymer and can be used as electron donor in methanol production. Methanol can be use as bio-fuel or external carbon source for denitrification to turn the WWTPs in to energy self-sufficient one. In addition, this process will increase nitrogen removal efficiency from main stream wastewater which is environment friendly and safe to public health. 3.5 Scope of Works It is very important to integrate methanotrophs in to WWTP process to mitigate AD generated biogas and recover valuable resource from it.The aim of this chapter is to report the performance of maintaining methanotrophic-heterotrophic culture maintained under wastewater mainstream conditions in fed-batch mode. Furthermore, the culture capacity to remove nitrogen either for cellular assimilation or nitrification-denitrification was explored. Thereafter, the active culture with same condition was used in batch experiment for methanol and Poly Hydroxyburate (PHB) production. 3.6 Materials and methods 3.6.1 Operational condition For enrichment and growth cycles, mineral salts medium (MSM) (Bowman, 2006) was used where the composition is as follows (in mg/L): MgSO4·7H2O, 1000; CaCl2·2H2O, 200; KH2PO4, 272; K2HPO4, 610; Fe-EDTA, 4 and 1 mL/L trace metal solution. The chemical composition of trace metal solution is (mg/L): ZnSO4·7H2O, 10; MnCl2·4H2O, 3; H3BO3, 30; Na2MoO4·2H2O, 3; FeSO4·7H2O, 200; NiCl2·6H2O, 2 and CoCl2·6H2O, 20. In addition, for all experiments copper sulfate (CuSO4·5H2O) concentration was made 10 μmol from a stock solution. Nitrate (in the form of NaNO3) and ammonium (in the form of NH4Cl) were used for enrichment and growth phase. 36 For all experiments’ incubation was done using 250-mL serum bottles (Wheaton, Mealville, NJ, USA) capped with butyl-rubber stoppers. Liquid volume was 50 mL, and the headspace volume was 200 mL. The headspace was evacuated by a suction pump for 5 minutes and then it was filled with oxygen and methane or helium gas (to maintain same pressure) with purity >99% (Praxair Technology, Inc., Danbury, CT, USA). Methane and oxygen were added with a volumetric ratio of 1:1. The bottles filled with liquid and gas were placed horizontally on a MaxQ™ 4000 Benchtop Orbital Shakers (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 165 rpm and the incubation temperature was 30℃. Gas and liquid sample measurements were carried out at the start and end of each cycle. For gas samples oxygen, nitrogen, methane and carbon dioxide were measured and for liquid samples optical density (OD600), COD concentration as mg/L, ammonium, nitrate, nitrite concentration as mg-N/L were measured. 3.6.2 Methanotrophs mixed culture enrichment In this stage methanotrophic mixed culture was enriched from the seed for the growth phase. The seed for cultivating methanotrophs cultures was collected from waste activated sludge from Humber wastewater treatment plant (Toronto, Canada). Fresh waste activated sludge from Humber wastewater treatment plant (Toronto, Canada) were used as a seed for type I methanotrophs enrichment. The sludge was filtered through 100-μm cell strainer to remove large particles. The filtered sludge was centrifuged and re-suspended in 50 ml MSM with 10 μM copper sulfate and 10 mM of sodium nitrate. Initial optical density (OD600) were kept to 0.5 ± 0.1 for four bottles. The headspace was filled with 200 mL of O2 and 200 mL of CH4 every 24 hours. In addition, the culture medium was changed with fresh medium by centrifuging (4200 rpm) for 20 mins every two days. After four days, cultures started to shift to a pinkish color known for type I methanotrophs. The enrichment was continued for fourteen days to get type I methanotrophs with 37 stable growth rate and gas consumption. Before starting of growth phase with ammonium and sodium acetate, the number of bottles increased to nine and liquid medium was changed every day with initial OD600 of 1 ± 0.1 to ensure the exponential phase. Samples are required for microbial analysis to confirm type I methanotrophs existence/dominance. 3.6.3 Growth Phase Enriched cultures were cultivated in four sets of experiment (table 1) in 50ml MSM with ammonium and COD. It was decided to maintain the ratio (w/w) of COD: NH4-N at around 6 for all sets of experiment. Sodium acetate was used to provide COD to liquid medium. For ammonium-nitrogen (NH4-N) concentration (mg/L) 30, 60 and 90 corresponding COD concentrations were (mg/L) 180, 360 and 540 respectively. 200 mL of O2 and 200 mL of CH4 were used for all four sets. For each set, bottles were triplicated for data consistency. The experiment was run in fed-batch for 50 consecutive cycles with 24 hr cycle duration. For first ten days all measurements were taken then for 17 days there were no measurement but for final 12 days all measurements were taken again. In each cycle initial OD600 was made to 2 ± 0.1, liquid medium was transferred with fresh medium and headspace was replaced with appropriate gas volume. 38 Table 3.1: Experimental conditions for fed-batch experiments Fed-Batch NH4+ COD (mg N/L) (mg /L) OD600 tests COD:N CH4 O2 (ml) (ml) Control 1 2.0 60 0 - 200 200 A 2.0 60 360 6:1 200 200 B 2.0 30 180 6:1 200 200 C 2.0 90 540 6:1 200 200 3.6.4 Analytical methods Gas samples were collected from serum bottles using an air tight syringe. With the same syringe, gas samples were injected in to a gas chromatography (SRI instrumentation, Torrance, USA) machine to analyse the gases. The GC is equipped with thermal conductivity detector (TCD), and molecular sieve column. The GC is connected to helium and hydrogen gas cylinder as carrier gas. The flow rate for helium and hydrogen gas were 15 ml/min and 20 ml/min respectively. GC analyses the gas through the column and gives peak of oxygen, nitrogen and methane consecutively at different time. With the peak area, gas concentration can be determined from previous calibration. In the program the temperature was set as: injector-80◦C; Oven- 80◦C; FID300◦C; TCD-155◦C. For optical density measurement, DR 3900 Benchtop Spectrophotometer (HACH Company, Loveland, Colorado, USA) was used. To obtain the dry cell weight (DCW), a previously developed correlation equation between OD600 and DCW was used (equation 1, alsayed et al, 2018). DCW (mg) = OD600 / 0.0021 X liquid volume (L)----------------------------------(1) 39 To measure the NH4+, NO3-, NO2- ion chromatograph (IC) was used. COD and total nitrogen were measured based on optical measurement by, HACH methods and testing kits. 3.7 Results and Discussion 3.7.1 Influence of varying COD on methanotrophic microbial activity The effect of COD on methanotrophic microbial activities tell us the possibility of growing methanotrophs under COD environment. The previously reported performance for methanotrophic activity was done in the presence of methanotrophs that favors the dominance and growth of methanotrophs. However, in real application in wastewater treatment plants, COD is expected with different concentrations based on the location of the methanotrophic reactor. The behavior of methanotrophs in COD environment with other microorganisms will be interesting. In this section, biomass microbial activities such as specific growth rate or µ (day-1), observed growth yield or Y (mg-VSS/mg-CH4), methane uptake rate (mg/hr), CH4 consumption, O2 consumption and COD consumption are reported from fifty cycles of operation under different COD concentrations. The calculations for these parameters are included in Appendix 1 and Appendix 2. Sodium acetate is used as COD source with concentration varying from 0 to 540 mg/L. Ammonia is used as nitrogen source with concentration varying from 30 to 90 mg/L with same initial biomass density of OD600 2.0±0.2 (952 mg/L). To reflect the main stream condition COD/N ratio is maintained as 6:1. For example, for 360 mg/L sodium acetate as COD concentration, nitrogen concentration is 60 mg/L as ammonia nitrogen and initial biomass density is OD600 2.08 ± 0.13. From previous studies (Ahmed AlSayed et al. 2018), optimum growth condition is maintained to achieve maximum biomass growth. Volume of added methane is 200 ml and volume of added oxygen is 200 ml to maintain 1:1 methane/oxygen ratio(v/v). As 40 mentioned earlier, all measurements had been taken for 10 cycles at the beginning and 12 cycles at the end and no measurements were taken for 28 cycles at the middle. Specific growth rate µ and biomass yield Y: As there are 22 cycles (10+12 cycles) data for different COD conditions, t-test were performed for data comparison between two conditions. In this study the data sets with varying COD were compared with data set with no COD. During t-test it was considered that all data were unequally variable. For t-test if p value is less than 0.05 then it can be considered that the data sets are different from each other. On the other hand, if p value is greater than 0.05 then the data sets are considered not significantly different. For first 10 cycles specific growth rate µ, biomass yield Y and methane uptake rate increased from no COD condition with increasing COD (Figure 1a). At COD 0 mg/L and NH4-N 60 mg /L average specific growth rate, µ and biomass yield, Y were 0.28±0.25 d-1, 0.30±0.25 mg-VSS/mgCH4 and 2.7±0.6 mg-CH4 /hr respectively. Maximum specific growth rate was 0.41±0.15 d-1 at COD 540 mg/L which is 1.46-fold (p=0.002) higher than no COD condition. The maximum value of observed yield and methane uptake rate were also found at COD 540 mg/L. The values are 0.36±0.14 mg-VSS/mg-CH4 and 3.2±0.5 mg-CH4 /hr respectively and they are 1.2-fold (p=0.002) and 1.18-fold (p=0.01) higher than no COD condition. The higher standard deviation was observed for the first 10 cycles due to the fluctuation of growth parameters at the beginning. After then the experiment kept running without any measurement till 36th cycle. All measurements were taken again from 37th till 50th cycle. For final 12 cycles, at no COD condition average specific growth rate and µ and biomass yield Y were 0.39±0.13 d-1 and 0.41±0.12 mg-VSS/mg-CH4 (Figure 1b) which are 1.39 (p=0.04) times and 1.36 (p=0.009) times higher than the respective values for first 10 cycles without COD condition (Figure 1b). Here less standard deviation than the beginning was 41 observed confirming the stable performance of the culture. The maximum µ and Y were found at COD 360 mg/L were 0.49±0.12 d-1 and 0.49±0.11 mg-VSS/mg-CH4 which are 1.25 (p=0.0008) times and 1.2 (p=0.0001) times higher than no COD condition. The values decreased marginally by 6 percent and 8 percent respectively at COD 540 mg/L. Which indicated that growth parameters increased till COD 360 mg/L after that growth may become stable or decline with increasing COD. To confirm this further experiment is required to perform with higher COD concentration for longer period. Methane uptake rate Another growth parameter, average methane uptake rate for first 10 cycles was found 2.7±0.3 mgCH4 /hr at no COD condition, which is 22% less (p=0.01<0.05) than the value at COD 540 mg/L. Here, p value is less than 0.05 signifies the difference in methane uptake rates for two COD conditions. For the final 12 cycles, methane uptake rate was 2.5±0.3 mg/hr, which remained unchanged (p=0.88>0.05) for increasing COD values. P value greater than 0.05 indicates no significant difference between two conditions. From this data it is evident that after 36 cycles methane uptake rate became stable and did not change with COD influence. This indicates that the COD has no significant influence on the methane utilization rate or methanotrophic activity of the consortium. Moreover, till certain level, COD had positive effect on the growth of the microbial community. Comparison with literature As mentioned earlier after day 36 most of the growth parameters were more stable than the beginning. Average values for all growth parameter from day 37 to day 50 are presented in table 1 to compare with literature values. Without COD the average specific growth rate was found 14 42 percent higher than the reported value. At no COD condition µ was 0.39±0.04d-1 where in literature (table 3.2) it is 0.344±0.06 for Type I methanotrophic mixed culture at OD 2.06 (AlSayed et al., 2018a). This confirms the best performance of the methanotrophic mixed culture at normal condition. The maximum µ at COD 360 mg/L is 1.44 times higher than the reported value by AlSayed et al. The Biomass Yield (mg-VSS/mg-CH4) for without COD is 0.41±.05 which is 33 percent higher than literature value of 0.33±0.04 for type I methanotrophs at OD 2.06 (AlSayed et al., 2018a). Average methane uptake rate (mg-CH4/hr) were 2.50±0.52, 2.53±0.60, 2.50±0.36 and 2.48 ±0.49 for 0, 180, 360 and 540 mg/L COD concentration which is to comparable to the literature value of 2.73mg-CH4/hr (AlSayed et al 2018). It was observed that the growth parameters showed increased values compared to reported values with the addition of COD. This might be due to the presence of other heterotrophic culture as COD favours the growth of heterotrophs. Overall, the incorporation of COD increased the specific growth rate, biomass yield and without affecting the methane utilization rate. 43 a: First 10 cycles b: Final 12 Cycles 0.6 0.5 0.4 0.28 0.3 0.41 0.39 0.36 Growth rate, µ Observed Yield, Y Growth rate, µ Observed Yield,Y 0.6 0.36 0.36 0.29 0.3 0.2 0.1 0.5 0.4 0.39 0.41 0.43 0.49 0.49 0.40 0.46 0.45 0.3 0.2 0.1 0.0 0.0 0 180 360 COD (mg/l) 0 540 180 360 COD (mg/l) 540 Avg Growth rate (day⁻1) obs Yield (g VSS/ g CH4) Avg Growth rate (day⁻1) obs Yield (g VSS/ g CH4) Figure 3.1: Average Growth rate (d-1), Methane Uptake rate (mg-CH4/hr) and Growth yield (g DCW/gCH4) at COD 0 mg/L, 180 mg/L, 360 mg/L and 540 mg/L for a) first 10 cycles b) final 12 cycles 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 2.7 2.9 3.0 b: Final 12 cycles 3.2 CH4 (mg/hr) CH4 (mg/hr) a: First 10 cycles 0 180 360 COD (mg/l) 540 Avg. CH₄ Uptake (mg/hr) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 2.5 2.5 2.5 2.5 0 180 360 COD (mg/l) 540 Avg. CH₄ Uptake (mg/hr) Figure 3. 2: Methane Uptake rate (mg-CH4/hr) at COD: 0 mg/L, 180 mg/L, 360 mg/L and 540 mg/L for a) first 10 cycles b) final 12 cycles 44 Table 3.2: Comparison of different growth parameters at different COD and ammonia concentration Parameter COD 0 (mg/l) 0.39 ±0.04 COD COD COD Literature Value 180 360 540 (for mixed culture without (mg/l) (mg/l) (mg/l) COD) 0.43 0.49 0.46 0.344±0.06 (Type I, at OD ±0.04 ±0.05 ±0.05 2.06) AlSayed et al 2018 Methane Uptake Rate (mg/hr) 2.50 ±0.52 2.53 ±0.60 2.50 ±0.36 2.48 ±0.49 2.73 (Type I, at OD 2.06) AlSayed et al 2018 Biomass Yield (mg-VSS/mgCH4) 0.41 ±0.05 0.40 ±0.05 0.49 ±0.04 0.45 ±0.04 0.33±0.04 (type I, at OD 2.06) AlSayed et al 2018 Specific Growth Rate (day-1) a: COD 0 mg/L, NH4-N 60 mg/L b: COD 180 mg/L, NH4-N 30 mg/L 100 80 CH4, O2 % CH4, O2 % 100 60 40 20 0 1 3 5 7 60 40 20 0 10 37 39 42 45 47 49 Cycle % CH4 Consumed 80 1 3 80 CH4, O2 % CH4, O2 % 100 60 40 20 0 7 60 40 20 0 10 37 39 42 45 47 49 1 Cycle % CH4 Consumed % O2 Consumed d: COD 540 mg/L, NH4-N 90 mg/L 80 5 10 37 39 42 45 47 49 % CH4 Consumed c: COD 360 mg/L, NH4-N 60 mg/L 3 7 Cycle % O2 Consumed 100 1 5 % O2 Consumed 3 5 7 10 37 39 42 45 47 49 Cycle % CH4 Consumed % O2 Consumed Figure 3. 3: Activity of biomass in terms of % CH4 Consumption and % O2 Consumption for a) COD 0 mg/L and ammonia-N 60 mg/L (control), b) COD 180 mg/L and ammonia-N 30 mg/L, c) COD 360 mg/L and ammonia-N 60 mg/L, d) COD 540 mg/L and ammonia-N 90 mg/L 45 For 22 cycles 100.0 CH4, O2 % 80.0 82.4 60.1 59.8 60.6 92.2 88.0 77.9 62.9 60.0 40.0 20.0 0.0 0 180 360 540 COD (mg/L) % CH4 Consumed % O2 Consumed Figure 3. 4: average percentage of CH4 and O2 consumption for different COD 3.7.2 Methane, Oxygen and COD consumption by the microbial community From methane and oxygen consumption data methanotrophic activity can be further confirmed. Moreover, COD consumption data ensures the growth of microorganisms other than methanotrophs. The calculation for all consumption data is attached in appendix I and II. The activity of the biomass monitored for different COD condition is compared without COD condition to reflect the influence of COD on the behaviour of the microbial community. For all COD condition methane and oxygen consumption was calculated from analytical data. Initially total methane and oxygen consumption percentage varied a little but after 10 cycles it became more consistent (Figure 3.3). For no COD condition, methane consumption varied from 45 to 73 percent and oxygen consumption varied from 65 to 88 percent (Figure 3.3 a). With COD 180 mg/L, methane and oxygen consumption became more stable after 36 cycles (Figure 3.3 b). From the beginning methane and oxygen consumption percentage varied from 43 to 70 percent and 67 to 89 percent respectively. With COD 360 mg/L there was fluctuation in methane consumption at the 46 beginning while oxygen consumption was more stable throughout the cycles (Figure 3.3 c). Methane consumption also varied from 81 to 72 percent while oxygen consumption varied less from 84 to 93 percent. Interestingly, for COD 540 mg/L methane and oxygen consumption percentage were more stable from the beginning compared to other conditions (Figure 3.3 d). The values ranged from 57 to 69 percent and 86 to 98 percent respectively. COD Consumption (mg/L), (%) a: For first 10 Cycles 600 463 500 400 267 300 200 97 86 74 54 100 0 180 360 COD (mg/L) COD Consumption (mg/l) 540 COD Consumption (%) b: For Final 12 cycles COD Consumption (mg/L), (%) 600 482 500 400 311 300 200 144 89 86 80 100 0 180 360 COD (mg/L) COD Consumption (mg/l) 540 COD Consumption (%) Figure 3. 5: Average COD Consumption as mg/L and percentage at COD 0 mg/L, 180 mg/L, 360 mg/L and 540 mg/L for a) first 10 cycles b) final 12 cycles 47 From the average values of 22 cycles data (figure 3.4) it was found that methane consumption was 60.1±10.1 percent for no COD condition. The value varied a little by increasing marginally by 5% at COD 540 mg/L. For other COD conditions the change in methane consumption was not significant. The oxygen consumption for no COD condition was 82.4±8.5 percent which was increased by maximum 12 percent at COD level 540 mg/L. Increasing COD had increased the oxygen consumption percentage. However, the oxygen consumption at COD 180 mg/L is 77.9±4 percent which is 2.5 percent less than no COD condition. This is might be the reason of low ammonia concentration for this condition. From the above data it is evident that the methanotrophic activity remains constant with COD incorporation but increasing oxygen consumption suggests the activity of heterotrophic bacteria with increasing COD concentration. COD consumption data are presented in two stages for first 10 cycles and then final 12 cycles due to data fluctuations at the beginning (Figure 3.5). For the first 10 cycles COD consumption increased with the increase of COD concentration. The maximum of 86±4 percent COD consumption was found at 540 mg/L COD. At COD 180 mg/L, 97±30 mg/L (54%) of COD consumed while the consumption was 267±26mg/l (74%) out of 360mg/L. The consumption further increased to 463 mg/L (86%) with higher COD of 540 mg/L. Similar trend but increased consumption was observed for the final 12 cycles with less standard deviation. Highest COD consumption was 482±20 mg/L (89%) at COD 540 mg/L and lowest was observed 144±18mg/L (80%) at 180 mg/L of COD. In this period the difference in COD consumption percentage from COD 180 mg/L to 540 mg/L is 8%which signifies the sustainability for longer period. The increase in COD consumption with inlet COD increase signifies that the activity or growth of heterotrophs is happening. As previously mentioned with the increase of COD, methane uptake rate was stable which meant methanotrophic bacterial activity was not changing but at the same time the COD 48 uptake rate was increasing with COD increase which means there is certainly some microorganisms other than methanotrophs present. 3.7.3 Nitrogen Removal To apply methanotrophs in nitrogen removal process in WWTP, it is necessary to confirm their nitrogen removing capability. Some studies reported that methanotrophs can partially denitrify under hypoxic condition (Stein and Klotz, 2011). For nitrogen removal, nitrification and denitrification are the major steps in conventional WWTPs. So, it is important to find out the nitrification and denitrification activities to confirm the possibility nitrogen removal by methanotrophs. Table 3.3: Average Ammonia consumption, nitrate, nitrite concentration at different COD level NO3- (mg/L) 14.6±19 % NH4-N Consumed 79.6±24 0.59±0.4 NO2(mg/L) 0 34.3±6.7 0.11±0.15 99.7±0.5 0.16±0.19 0 360 60.8±10.7 0.16±0.29 99.72±0.48 0.27±0.23 0 540 91.5±5.6 2.3±3.4 97.45±3.5 0.12±0.1 0 COD (mg/L) 0 NH4-N In (mg/L) 61.6±10 NH4-N Out (mg/L) 180 To monitor the nitrogen removal, ammonia nitrogen, nitrate and nitrite were measured at the end of each cycle using Ion Chromatograph (IC). All calculation is attached in appendix II. The set without COD showed fluctuations at the beginning but from cycle 38 it showed consistent results. The average percentage of ammonia consumption was 79.6±24 percent (Table 2). Microbial community with COD showed consistent removal of ammonia from the beginning. For COD 180 and 360 mg/L the ammonia consumption was 99.7±0.5 percent and 99.72±0.48 percent and for COD 90 mg/L the value was 97.45±3.5 percent. So, from these data it can be said that the addition of COD had not much effect on ammonia consumption. There is little bit less ammonia 49 consumption at COD 90 mg/L which might be the toxic effect of hydroxylamine from ammonia. In addition, there was an insignificant amount of nitrate and no nitrite found at the end of each cycle. This confirms that the nitrogen might be used for cell synthesis or might be converted to gas by denitrification. To confirm this, it is required to take samples at short interval to see any nitrate or nitrite is present or not, also to measure the nitrogen and nitric oxide gas to see if there is any denitrification. In addition, from the nitrogen balance it can be said that how much nitrogen is used for cell synthesis and how much is converted to gas. 3.7.4 Nitrogen Balance The purpose of nitrogen balance is to determine how much nitrogen is going inside the system and how much nitrogen is left after the experiment. In addition, to determine the percentage of nitrogen inside the biomass and finally to determine how much nitrogen is converted to gas. 3.7.4.1 Methods For nitrogen balance samples including biomass and liquid feed were collected at the end of each cycle and stored in a fridge. Then total nitrogen test was performed for selected samples using HACH vials (Method 10071). From the test total nitrogen (out) for each sample was measured and then total nitrogen (in) was calculated by back calculation. For this inorganic nitrogen (NH4, NO3, NO2) in the liquid sample was also measured by ion chromatograph (IC). Then organic nitrogen was calculated by subtracting inorganic nitrogen from total nitrogen. Then percentage of nitrogen in biomass was calculated by dividing the organic nitrogen by dry cell weight (DCW). After then, total nitrogen (in) using nitrogen percentage in cell, DCW (in) and inorganic nitrogen (in). Finally, the amount of nitrogen converted to gas was calculated from in and out total nitrogen data. Inorganic Nitrogen (INout) = NH4 (mg/L) + NO3 (mg/L) + NO2 (mg/L) 50 Organic Nitrogen (ONout)= TNout (mg/L) - INout (mg/L) % of N in Biomass= ONout (mg/L)÷ DCWOut(mg/L) TN In= NH4-NIn + (DCWIn x % of N in biomass) Nitrogen converted to gas (mg/L) = TNIn - TNout 3.7.4.2 Result and discussion 200 200 165 N (mg/L) 150 132 99 100 50 170 153 146 86 19 30 19 12 0 0 180 360 540 COD (mg/L) Total Nitrogen IN (mg/l) NitrogenConverted to gas (mg/l) Total Nitrogen OUT (mg/l) Figure 3. 6: Nitrogen balance data at different COD concentration showing In an Out of total nitrogen and amount of nitrogen converted to gas Nitrogen converted to gas From nitrogen balance data (Figure 3) for each condition the total nitrogen (out) was less than the total nitrogen (in). Which confirmed that some nitrogen is converted to gas. At COD 0 and 360 mg/L the NH4-N was 60 mg/L, which shows almost similar nitrogen conversion to gas of 19 mg/L but the later one showed 1% higher nitrogen gas conversion. From COD 180 to 540 mg/L NH4-N was increased form 30 to 90 mg/L where in and out data for total nitrogen were increased proportionately. In addition, the amount of nitrogen converted to gas was increased from 12 to 30 51 mg/L. The percentages of total nitrogen gas conversion were increased with increased COD concentration. The values were 11.5, 12.1, 12.4 and 15 percent for COD 0, 180, 360 and 540 mg/L respectively. So, COD along with NH4-N concentration increase caused higher nitrogen gas conversion. During nitrogen balance, the percentage of nitrogen in biomass was calculated. It was observed that the percentage was varied from 8 to 11 percent (Figure 4). The lowest nitrogen in biomass was observed at NH4-N 30 mg/L and the highest nitrogen content was observed at NH4N 90 mg/L. With same NH4-N 60 mg/L at COD 0 and 360 mg/L shows the nitrogen percentage is 1 percent less with incorporation of COD. Theoretically, bacteria cell contains 12 percent of nitrogen considering the chemical formula C5H7O2N. From the data it can be said that the nitrogen percentage at higher ammonia concentration is close to theoretical value. % N in Biomass %NH4 to Gas and Biomass 80 56 70 64 61 57 60 50 36 37 40 34 33 30 20 11 11 10 8 10 0 0 180 360 540 COD (mg/L) % N in biomass % NH4 to Gas % NH4 to biomass Figure 3. 7: Ammonia utilization pathway showing the percentage of ammonia (in) converted to gas, utilized for cell synthesis and percentage of nitrogen inside the biomass Ammonia utilization pathway From amount of total nitrogen converted to gas and organic nitrogen in biomass data how much ammonia nitrogen from feed was converted to nitrogen gas and utilized in the cell was calculated. 52 Table 3.4: Ammonia utilization pathways at different COD condition COD (mg/L) 0 NH4-N in (mg/L) 60 % NH4-N to gas 33.4 % NH4-N to Biomass 56.2 % NO3-, NO21.16 % Unused NH4 9.3 180 30 37.4 61.4 0.5 0.6 360 60 36.0 63.6 0.3 0.1 540 90 34.4 57.1 0.44 8.1 With the increase of ammonia concentration from 30 to 60 mg/L % NH4-N to biomass conversion increased from 61.4 to 63.6 percent but NH4-N at 90 mg/L the conversion percentage reduced by 3 percent. In addition, about 8 percent NH4-N remained unused at this concentration. This can be explained as, at higher level of ammonia the toxic effect may inhibited the biomass growth. The maximum ammonia to gas conversion was observed at NH4-N 30 mg/L which signify that low level of ammonia is favourable for gas conversion. There was 9.3 percent ammonia remained unused at no COD and NH4-N 60 mg/L while with COD 360 and same ammonia the almost all ammonia was utilized by the bacteria and the nitrogen conversion was increased by 3 percent from 33.4 percent. This implies that COD addition has positive impact on the microbial community in nitrogen utilization and removal. 3.8 Conclusion Addition of COD positively impacted the growth of methanotrophic mixed culture in terms of increased growth activities of growth rate and growth yield. Moreover, methane uptake rate by the consortium remained unchanged with addition of COD which signifies the stable and sustainable behavior of methanotrophs under COD condition. In addition, COD consumption by the consortium indicate the presence of other microorganism which also supports the fact of surviving 53 of methanotrophs with other cultures. From nitrogen balance data, it was found that some part of consumed nitrogen is being converted towards gaseous form of nitrogen in addition to cell synthesis. This data supports the denitrification by methanotrophic mixed consortium in wastewater treatment process. Finally, methanotrophs can be grown in mainstream condition with production of value-added product and nitrogen removal from wastewater. 54 CHAPTER 4: Methanol and PHB production by Methanotrophicheterotrophic mixed culture performance under different COD concentrations 4.1 Introduction ‘Methanotrophs’ have the ability to convert methane in to biofuel like methanol and biopolymer like PHB. Methanol has an higher energy density than methane and can be used as alternate fuel for cars and does not have safety issues with storage and transportation (Hwang et al., 2014). Similarly, PHB is a eco friendly biopolymer which is degradable and can be recycled and restocked to the environment. Methanotrophs oxidize methane aerobically into methanol which is further oxidized terminally into carbon dioxide gas through a series of intermediated like formaldehyde and formate (Ge et al., 2014). Usually, methanol production is done by inhibiting the MDH enzyme which is responsible for methanol oxidation. For inhibition phosphate and sodium chloride are used and formate is used as external electron source for survival and metabolic activity of methanotrophs (Ge et al., 2014). On the other hand, certain type of methanotrophs can produce PHB as intracellular granules under nutrient limiting condition. In nutrient limiting condition such as without nitrogen, methanotrophs adopts survival strategy and switch themselves to PHB cycle to provide energy for cellular maintenance (Karthikeyan et al., 2015). The integration of methanol and PHB production into WWTPs have many challenges like: grow methanotrophs inside the system, utilize methanotrophs for treatment processes and produce valuable product using the feed from mainstream WW. In chapter 3, it is shown that methanotrophs can grow in mainstream WW condition, but is it possible to use the same condition for application side? To answer this question two applications of methanotrophs were done by main stream WW 55 condition. To do that like as growth phase in previous chapter, ammonia and COD with varying concentration were applied to methanol and PHB production consecutively. 4.2 Materials and Methods 4.2.1 Methanol production After 50 cycles of methanotrophs growth phase with COD the same biomass was collected and enriched with same liquid and gas feed for methanol batch experiment. The enrichment continued until stable activity in terms of growth rate, methane and oxygen consumption was achieved. Methanol batch experiment was carried by 6 hours growth phase followed by 4 hours methanol phase with different liquid medium. 4.2.2 Growth phase The enriched active biomass was used for growth phase with initial OD ~3.0 in 50 ml liquid medium where MSM, trace metal, copper, ammonium and COD were added. The ingredients for MSM were: MgSO4·7H2O (1000 mg/L), CaCl2·2H2O (200 mg/L), KH2PO4 (272 mg/L), K2HPO4 (610 mg/L), Fe-EDTA (4 mg/L) in addition to this 1 mL/L trace metal solution and 1.6 mg/L copper sulfate (CuSO4·5H2O) were added. For the batch four different sets were used with different nitrogen and and COD concentration. For nitrogen source NH4Cl was used and for COD sodium acetate (CH₃COONa) was used. For four sets to make 0, 180, 360, 540 mg/L COD solution, 0, 225, 400 and 600 mg/L CH₃COONa were added respectively. To make 30, 60 and 90 mg/L NH4-N solution, 114.5, 229 and 343.5 mg/L NH4Cl added respectively. For each condition of experiment, triplicated 250 ml serum bottles were used for consistency in results. After suspension of the biomass in liquid medium, each bottle was evacuated for 5 minutes using a suction pump. Afterwards, the bottles were fed with 200 ml methane and 200 ml oxygen 56 gas and all the bottles were incubated in a orbital shaker for 6 hr at a speed of 165 rpm and temperature of 25 to 30℃. At the end of growth phase all biomass in each bottle was collected after centrifuging them. 4.2.3 Methanol Phase Biomass from the growth phase was used for methanol production. The liquid feed was changed for MSM with MDH inhibitor and electron donor. In 50 ml solution the following chemicals were used as inhibitors: MgCl2.6H2O (2033 mg/L), NaH2PO4 (2879.5 mg/L), Na2HPO4 (9311.5 mg/L), electron donor: Sodium Formate (8160 mg/L). In addition, 1ml/L trace metal from stock solution, 1.6 mg/L CuSO4·5H2O and ammonia and COD concentration were the same as growth phase. Same methane and oxygen gas were used as growth phase and the samples were incubated in the orbital shaker for 4 hours. Then all the samples were centrifuged to collect supernatant for methanol measurement. The supernatant was injected in a GC equipped with a MXT-WAX column where a flame ionization detector (FID) was used. The injector oven temperature was 30℃ and FID temperature was 300℃ and helium was used as the carrier gas. 4.2.4 PHB production After methanol batch experiment all biomass were collected and enriched again with MSM, trace, copper, ammonia and COD. The enrichment continued in four different sets till the biomass activity became stable in terms of growth rate, methane and oxygen consumption. Experimental condition for PHB Enriched biomass was used for PHB batch experiment with initial OD 1.5~2.0 in four different sets. For liquid feed MSM, trace metal and copper were used same as before, but no nitrogen source was used to create N-limiting condition. In addition, to incorporate COD, 0, 225, 400 and 57 600 mg/L CH₃COONa were added in for different sets. At the beginning of the experiment 200 ml of oxygen and 200 ml of methane were injected to each serum bottle which were replaced with same gases after 24 hours. All the bottles fed with biomass were incubated in the orbital shaker at 165 rpm and 30℃ for 48 hours. After the experiment, the PHB enriched biomass were collected for measurement. Analytical Measurements The PHB was measured by the method developed by Braun egg, Sonnleitner and Lafferty (Braunegg et al., 1978). At the beginning of this process, 10−15 mg of biomass sample is collected in a test tube where 2 mL of acidified methanol (3% sulfuric acid) and 2 mL of chloroform are added to a glass vial. The mixture is then subjected to heat at 100 °C for 3.5 h and then left to cool down to room temperature. After then, 1 mL of deionized water is added to the mixture and vortexed for 1 min, and then kept for a while for phase separation. The lower organic phase is tested for PHB measurement using a gas chromatograph equipped with a flame ionization detector (FID) and an MXT-wax column. The temperature condition is: 1 min 80 °C, 10 °C/min, 180 °C for 4 min. The results are calibrated with standard curves obtained using PHB standards (Sigma-Aldrich). Benzoic acid is used as an internal standard to increase the accuracy. 58 4.3 Results and Discussion 4.3.1 Influence of COD on methane and oxygen consumption during methanol production % CH4, O2 Consumed 60 42 40 25 21 20 14 19 9 8 2 0 0 180 360 540 COD (mg/L) % O₂ Consumed (Methanol) % CH₄ Consumed(Methanol) Figure 4.1: Percentage of CH4 and O2 Consumption during methanol production phase During methanol production batch experiment low methane and oxygen consumption were observed (Figure 4.1) which is a usual case as MDH inhibitors inhibit both MDH and MMO activity as well (Ge et al., 2014). At no COD condition 21 % oxygen and 14% methane was consumed whereas with same ammonia and with 360 mg/L COD resulted increased oxygen consumption of 25% but decreased methane consumption of 8%. Here the addition of COD somehow inhibiting the methane consumption which means methanotrophic activity during methanol production might be hampered by COD addition. Moreover, With the increase of COD from 180 to 540 mg/L the oxygen consumption increased from 9% to 42 % and methane consumption increased from 2% to 19%. So, both oxygen and methane consumption increased with the increase of COD concentration. From this data it can be interpreted that increasing COD 59 has a positive effect on methane and oxygen consumption, but more investigation is required to determine highest COD at which maximum methanotrophic activity will occur. % CH4, O2 Consumed 4.3.2 Influence of COD on methane and oxygen consumption during PHB production 90 80 70 60 50 40 30 20 10 0 78 55 50 36 24 33 29 15 0 180 360 540 COD (mg/L) % O₂ Consumed (PHB) % CH₄ Consumed(PHB) Figure 4. 2: Methane and Oxygen consumption Percentage during PHB production During PHB production with N-limiting condition methane and oxygen consumption were higher than methanol production phase. Moreover, higher methane and oxygen consumption were observed with the addition of COD compared to no COD where ammonia nitrogen were 60 mg/L for both cases. Oxygen consumption increased from 29% to 78% and methane consumption increased from15% to 55% at COD increase from 180 mg/L to 540mg/L, respectively. So, in Nlimiting condition COD has a positive effect on methanotrophic activity. 4.3.3 Influence of COD on methanol and PHB production Under desirable condition, methanotrophs are able to produce methanol and PHB. However, it would be interesting to see the ability of methanotrophs to produce methanol and PHB under COD 60 environment. In the batch experiment, methanol and PHB production were examined under varying COD. Table 4. 1: Methanol and PHB production at different COD condition COD (mg/L) Methanol (mg/L) PHB % 0 91±11 24±5 180 0 0 360 19±4 4±1 540 63±6 0 Without COD and with ammonia, methanol production is 91 mg/L (Table 4.1) which is 70 percent less than the reported methanol production with nitrate as nitrogen source (AlSayed et al., 2018d). No methanol was detected at COD 180 mg/L and NH4-N 30 mg/L while with the increase of COD to 360 mg/L and NH4-N 60 mg/L methanol production increases to 19±4 mg/L. With COD maximum methanol production was found 63±6 mg/L at COD 540 mg/L and NH4-N 60 mg/L.For PHB production at no COD condition PHB production is 24±5 percent which is 25 percent lower than the reported value for methanotrophic mixed culture(Fergala et al., 2018a). The lower production of PHB might be due to dominance of type I methanotrophs. PHB yield can be increased by providing proper condition to select type II methanotrophs. Unfortunately, with the addition of COD PHB production negatively impacted. Only 4 % of PHB production detected at COD 360 mg/L and NH4-N 60 mg/L. To maximize the PHB production, better understanding of the behavior of PHB producing bacteria under COD condition is required. 61 CHAPTER 5: Conclusion and Future Work 5.1 Conclusion There is no doubt for application for methanotrophs in methane mitigation and valuable resource recovery. It would be a revolution if this biotechnical application can be integrated in to WWTP. From the study, it is evident that methanotrophs can grow in mainstream condition, but it is necessary to decide where to put them in existing treatment processes like: to grow methanotrophs in the aerobic reactor with biogas generated from AD. The summary of the thesis findings are as follows: • From 50 cycles activities of the biomass in terms of specific growth rate, growth yield and methane uptake rate in different COD condition, it is evident that methanotrophs can survive in COD environment with other microorganism like heterotrophic bacteria. • From stoichiometric data, it is visible that the biomass activity became stable after 36 cycle of activity. Highest activity of the microbial community in terms of cellular growth and methane and COD removal was found at COD 360 mg/l. These findings signify that in COD condition, it takes about 36 days to the methanotrophic microbial consortium to become stable. Moreover, the best microbial performance at COD 360 mg/L confirms the applicability of methanotrophs in domestic WW effluent which contains about 300-500 mg/L of COD. • Average maximum COD consumption was 89±5 percent at COD 540mg/L and the average for ammonia consumption was around 99.72±0.48 percent at COD 360 mg/L. As 60 percent of domestic sewage contains ammonia, methanotrophs are able to remove ammonia from domestic WW. 62 • Addition of COD is favourable for heterotrophic bacteria growth without affecting methanotrophic bacterial activity. From this finding, it can be said that methanotrophs are able to survive in diverse microbial community. • COD consumption was increased proportionately with the increase of COD concentration till 540 mg/L means the activity of heterotrophic bacteria is positively affected in presence of methanotrophs and the COD removal capacity also increases with the influent COD increase. • Addition of ammonia up to 60 mg/L did not affected the ammonia consumption while ammonia concentration at 90 mg/L the consumption was decreased by 2.3 percent. Due to the toxicity of ammonia, methanotrophic activity might be hampered at ammonia level of 90 mg/L. Usually, domestic WW contains 20 to 60 mg/L of total nitrogen which still remains suitable to methanotrophic bacterial growth • From nitrogen balance, it was observed that 19 mg/L (12.5%) of nitrogen from 153 mg/L of total nitrogen was converted to gaseous nitrogen at NH4-N and COD concentration 60 mg/L and 360 mg/L respectively. With the increase of ammonia up to 360 mg/L, the nitrogen gas conversion increases. • Percentage of nitrogen inside biomass was found 11% at 90 mg/L of NH4-N which is close to the theoretical value of 12% nitrogen content in biomass. • 100% ammonia was utilized at 60 mg/L NH4-N level from which 64% ammonia was converted to gaseous nitrogen and 36% was consumed into biomass. • Maximum methanol production was found 63 mg/L at COD 540 mg/L which is 30% less than without COD condition and 70% less than reported value for type I methanotrophs. 63 Without Cod less methanol was observed than reported value, the reason might be the presence of both type I and II, as type I methanotrophs can yield more methanol. In COD much less methanol yield was observed which signify the negative impact of COD on methanol production. • Only 4 % of PHB production detected at COD 360 mg/L, no PHB was detected in COD 180 and 540 mg/L. PHB production was significantly impacted by the addition of COD. To increase PHB yield, certain conditions require to increase the percentage of type II methanotrophs in the consortium. 64 5.2 Future Study • In future, the culture can be grown in a continuous system with reduced SRT to produce more solids to reproduce biogas. • For methanol production further study can be done with more ammonia and COD loading rate to maximize methanol yield. • To maximize PHB production, it is required to study the behavior of PHB producing methanotrophs (type II) in mainstream condition. Moreover, to increase the number of PHB producing type II bacteria, various techniques such as optimizing ammonia concentration or by feast and famine approach can be applied during growth phase. • After growing methanotrophs in mainstream condition, the biomass can be applied to methanol and PHB production with WW effluent or AD centrate as feed to minimize the COD effect. 65 BIBLIOGRAPHY Ahansazan, B., Afrashteh, H., Ahansazan, N., Ahansazan, Z., 2014. Activated Sludge Process Overview. Int. J. Environ. Sci. Dev. 5, 5. Al-Omari, A., Wett, B., Nopens, I., De Clippeleir, H., Han, M., Regmi, P., Bott, C., Murthy, S., 2015. Model-based evaluation of mechanisms and benefits of mainstream shortcut nitrogen removal processes. Water Sci. Technol. J. Int. Assoc. Water Pollut. Res. 71, 840–847. https://doi.org/10.2166/wst.2015.022 AlSayed, A., Fergala, A., Eldyasti, A., 2018a. Influence of biomass density and food to microorganisms ratio on the mixed culture type I methanotrophs enriched from activated sludge. J. Environ. Sci. 70, 87–96. https://doi.org/10.1016/j.jes.2017.11.017 AlSayed, A., Fergala, A., Eldyasti, A., 2018b. Sustainable biogas mitigation and value-added resources recovery using methanotrophs intergrated into wastewater treatment plants. Rev. Environ. Sci. Biotechnol. 17, 351–393. https://doi.org/10.1007/s11157-018-9464-3 AlSayed, A., Fergala, A., Khattab, S., Eldyasti, A., 2018c. Kinetics of type I methanotrophs mixed culture enriched from waste activated sludge. Biochem. Eng. J. 132, 60–67. https://doi.org/10.1016/j.bej.2018.01.003 AlSayed, A., Fergala, A., Khattab, S., ElSharkawy, A., Eldyasti, A., 2018d. Optimization of methane biohydroxylation using waste activated sludge mixed culture of type I methanotrophs as biocatalyst. Appl. Energy 211, 755–763. https://doi.org/10.1016/j.apenergy.2017.11.090 Babel, W., 1992. Pecularities of methylotrophs concerning overflow metabolism, especially the synthesis of polyhydroxyalkanoates. FEMS Microbiol. Lett. 103, 141–148. https://doi.org/10.1111/j.15746968.1992.tb05831.x Bachmann, N., n.d. Sustainable biogas production in municipal wastewater treatment plants. Wastewater Treat. 20. Barik, D., Murugan, S., 2014. Investigation on combustion performance and emission characteristics of a DI (direct injection) diesel engine fueled with biogas–diesel in dual fuel mode. Energy 72, 760– 771. https://doi.org/10.1016/j.energy.2014.05.106 Birth, T., Heineken, W., He, L., 2014. Preliminary design of a small-scale system for the conversion of biogas to electricity by HT-PEM fuel cell. Biomass Bioenergy, 21st European Biomass Conference 65, 20–27. https://doi.org/10.1016/j.biombioe.2014.04.002 Bodík, I., Sedláček, S., Kubaská, M., Hutňan, M., 2011. Biogas Production in Municipal Wastewater Treatment Plants – Current Status in EU with a Focus on the Slovak Republic. Chem. Biochem. Eng. Q. 25, 335–340. Bogner, J., Pipatti, R., Hashimoto, S., Diaz, C., Mareckova, K., Diaz, L., Kjeldsen, P., Monni, S., Faaij, A., Gao, Q., Zhang, T., Ahmed, M.A., Sutamihardja, R.T.M., Gregory, R., Intergovernmental Panel on Climate Change (IPCC) Working Group III (Mitigation), 2008. Mitigation of global greenhouse gas emissions from waste: conclusions and strategies from the Intergovernmental Panel on Climate Change (IPCC) Fourth Assessment Report. Working Group III (Mitigation). Waste Manag. Res. J. Int. Solid Wastes Public Clean. Assoc. ISWA 26, 11–32. https://doi.org/10.1177/0734242X07088433 Bowman, J., 2006. The Methanotrophs — The Families Methylococcaceae and Methylocystaceae, in: Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H., Stackebrandt, E. (Eds.), The Prokaryotes. Springer New York, New York, NY, pp. 266–289. https://doi.org/10.1007/0-38730745-1_15 Bowman, J.P., 2014. The Family Methylococcaceae, in: Rosenberg, E., DeLong, E.F., Lory, S., Stackebrandt, E., Thompson, F. (Eds.), The Prokaryotes: Gammaproteobacteria. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 411–440. https://doi.org/10.1007/978-3-642-38922-1_237 Braunegg, G., Sonnleitner, B., Lafferty, R.M., 1978. A rapid gas chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass. Eur. J. Appl. Microbiol. Biotechnol. 6, 29–37. https://doi.org/10.1007/BF00500854 66 Brock Biology of Microorganisms, 14th Edition [WWW Document], n.d. URL http://www.mypearsonstore.ca/bookstore/brock-biology-of-microorganisms-0321897390 (accessed 6.23.19). Budzianowski, W.M., 2016. A review of potential innovations for production, conditioning and utilization of biogas with multiple-criteria assessment. Renew. Sustain. Energy Rev. 54, 1148– 1171. https://doi.org/10.1016/j.rser.2015.10.054 Budzianowski, W.M., 2010. Negative Net CO2 Emissions from Oxy-Decarbonization of Biogas to H2. Int. J. Chem. React. Eng. 8. https://doi.org/10.2202/1542-6580.2455 Budzianowski, W.M., Budzianowska, D.A., 2015. Economic analysis of biomethane and bioelectricity generation from biogas using different support schemes and plant configurations. Energy 88, 658–666. https://doi.org/10.1016/j.energy.2015.05.104 Cantera, S., Lebrero, R., García-Encina, P.A., Muñoz, R., 2016. Evaluation of the influence of methane and copper concentration and methane mass transport on the community structure and biodegradation kinetics of methanotrophic cultures. J. Environ. Manage. 171, 11–20. https://doi.org/10.1016/j.jenvman.2016.02.002 Chai, C., Zhang, D., Yu, Y., Feng, Y., Wong, M.S., 2015. Carbon Footprint Analyses of Mainstream Wastewater Treatment Technologies under Different Sludge Treatment Scenarios in China. Water 7, 918–938. https://doi.org/10.3390/w7030918 Chen, S., Harb, M., Sinha, P., Smith, A.L., 2018. Emerging investigators series: revisiting greenhouse gas mitigation from conventional activated sludge and anaerobic-based wastewater treatment systems. Environ. Sci. Water Res. Technol. 4, 1739–1758. https://doi.org/10.1039/C8EW00545A Chistoserdova, L., Vorholt, J.A., Lidstrom, M.E., 2005. A genomic view of methane oxidation by aerobic bacteria and anaerobic archaea. Genome Biol. 6. Cicerone, R.J., Oremland, R.S., 1988. Biogeochemical aspects of atmospheric methane. Glob. Biogeochem. Cycles 2, 299–327. https://doi.org/10.1029/GB002i004p00299 Corradini, A., Mccormick, J., 2010. Process and System for Converting Biogas to Liquid Fuels. WO2010078035 (A2). Costa, C., Vecherskaya, M., Dijkema, C., Stams, A.J., 2001. The effect of oxygen on methanol oxidation by an obligate methanotrophic bacterium studied by in vivo 13C nuclear magnetic resonance spectroscopy. J. Ind. Microbiol. Biotechnol. 26, 9–14. CRI - Carbon Recycling International [WWW Document], n.d. . CRI - Carbon Recycl. Int. URL https://www.carbonrecycling.is (accessed 8.23.19). Criddle, C.S., Sundstrom, E.R., 2015. Intermittent application of reduced nitrogen sources for selection of PHB producing methanotrophs. US20150159185A1. Culpepper, M.A., Rosenzweig, A.C., 2012. Architecture and active site of particulate methane monooxygenase. Crit. Rev. Biochem. Mol. Biol. 47, 483–492. https://doi.org/10.3109/10409238.2012.697865 Czepiel, P.M., Crill, P.M., Harriss, R.C., 1993. Methane emissions from municipal wastewater treatment processes. Environ. Sci. Technol. 27, 2472–2477. https://doi.org/10.1021/es00048a025 Daims, H., Lücker, S., Wagner, M., 2016. A New Perspective on Microbes Formerly Known as NitriteOxidizing Bacteria. Trends Microbiol. 24, 699–712. https://doi.org/10.1016/j.tim.2016.05.004 Dam, B., Dam, S., Blom, J., Liesack, W., 2013. Genome Analysis Coupled with Physiological Studies Reveals a Diverse Nitrogen Metabolism in Methylocystis sp. Strain SC2. PLoS ONE 8. https://doi.org/10.1371/journal.pone.0074767 Di Maria, F., Micale, C., Sordi, A., 2014. Electrical energy production from the integrated aerobicanaerobic treatment of organic waste by ORC. Renew. Energy 66, 461–467. https://doi.org/10.1016/j.renene.2013.12.045 Donner, L., Ramanathan, V., 1980. Methane and Nitrous Oxide: Their Effects on the Terrestrial Climate. J. Atmospheric Sci. 37, 119–124. https://doi.org/10.1175/15200469(1980)037<0119:MANOTE>2.0.CO;2 67 El-Fadel, M., Massoud, M., 2001. Methane emissions from wastewater management. Environ. Pollut. Barking Essex 1987 114, 177–185. Farhad, S., Hamdullahpur, F., Yoo, Y., 2010. Performance evaluation of different configurations of biogas-fuelled SOFC micro-CHP systems for residential applications. Int. J. Hydrog. Energy 35, 3758–3768. https://doi.org/10.1016/j.ijhydene.2010.01.052 Fergala, A., AlSayed, A., Eldyasti, A., 2018a. Behavior of type II methanotrophic bacteria enriched from activated sludge process while utilizing ammonium as a nitrogen source. Int. Biodeterior. Biodegrad. 130, 8–16. https://doi.org/10.1016/j.ibiod.2018.03.010 Fergala, A., AlSayed, A., Khattab, S., Ramirez, M., Eldyasti, A., 2018b. Development of MethaneUtilizing Mixed Cultures for the Production of Polyhydroxyalkanoates (PHAs) from Anaerobic Digester Sludge. Environ. Sci. Technol. 52, 12376–12387. https://doi.org/10.1021/acs.est.8b04142 Fernández, I., Dosta, J., Mata-Álvarez, J., 2016. A critical review of future trends and perspectives for the implementation of partial nitritation/anammox in the main line of municipal WWTPs. Desalination Water Treat. 57, 27890–27898. https://doi.org/10.1080/19443994.2016.1235152 Ge, X., Yang, L., Sheets, J.P., Yu, Z., Li, Y., 2014. Biological conversion of methane to liquid fuels: Status and opportunities. Biotechnol. Adv. 32, 1460–1475. https://doi.org/10.1016/j.biotechadv.2014.09.004 Ge, Z., Zhang, F., Grimaud, J., Hurst, J., He, Z., 2013. Long-term investigation of microbial fuel cells treating primary sludge or digested sludge. Bioresour. Technol. 136, 509–514. https://doi.org/10.1016/j.biortech.2013.03.016 Gilbert, E.M., Agrawal, S., Karst, S.M., Horn, H., Nielsen, P.H., Lackner, S., 2014. Low Temperature Partial Nitritation/Anammox in a Moving Bed Biofilm Reactor Treating Low Strength Wastewater. Environ. Sci. Technol. 48, 8784–8792. https://doi.org/10.1021/es501649m Gunnerman, R.W., Gunnerman, P.W., 2009. Conversion of Biogas to Liquid Fuels. US2009250330 (A1). Gunsalus, N.J., Koppaka, A., Park, S.H., Bischof, S.M., Hashiguchi, B.G., Periana, R.A., 2017. Homogeneous Functionalization of Methane. Chem. Rev. 117, 8521–8573. https://doi.org/10.1021/acs.chemrev.6b00739 Hamad, T.A., Agll, A.A., Hamad, Y.M., Bapat, S., Thomas, M., Martin, K.B., Sheffield, J.W., 2014. Study of combined heat, hydrogen and power system based on a molten carbonate fuel cell fed by biogas produced by anaerobic digestion. Energy Convers. Manag. 81, 184–191. https://doi.org/10.1016/j.enconman.2014.02.036 Hanson, A.M., Lee, G.F., 1971. Forms of organic nitrogen in domestic wastewater. J. - Water Pollut. Control Fed. 43, 2271–2279. Hanson, R.S., Hanson, T.E., 1996. Methanotrophic bacteria. Microbiol Mol Biol Rev 60, 439–471. Hasan, M.M.F., Zheng, A.M., Karimi, I.A., 2009. Minimizing boil-off losses in liquefied natural gas transportation. Scopus. He, R., Chen, M., Ma, R.-C., Su, Y., Zhang, X., 2017. Ammonium conversion and its feedback effect on methane oxidation of Methylosinus sporium. J. Biosci. Bioeng. 123, 466–473. https://doi.org/10.1016/j.jbiosc.2016.11.003 Henckel, T., Roslev, P., Conrad, R., 2000. Effects of O2 and CH4 on presence and activity of the indigenous methanotrophic community in rice field soil. Environ. Microbiol. 2, 666–679. https://doi.org/10.1046/j.1462-2920.2000.00149.x Ho, A., Vlaeminck, S.E., Ettwig, K.F., Schneider, B., Frenzel, P., Boon, N., 2013. Revisiting Methanotrophic Communities in Sewage Treatment Plants. Appl. Environ. Microbiol. 79, 2841– 2846. https://doi.org/10.1128/AEM.03426-12 Hoefman, S., van der Ha, D., Boon, N., Vandamme, P., De Vos, P., Heylen, K., 2014. Niche differentiation in nitrogen metabolism among methanotrophs within an operational taxonomic unit. BMC Microbiol. 14, 83. https://doi.org/10.1186/1471-2180-14-83 68 Höglund-Isaksson, L., 2012. Global anthropogenic methane emissions 2005&ndash;2030: technical mitigation potentials and costs. Atmospheric Chem. Phys. 12, 9079–9096. https://doi.org/10.5194/acp-12-9079-2012 Holmen, A., 2009. Direct conversion of methane to fuels and chemicals. Catal. Today 142, 2–8. https://doi.org/10.1016/j.cattod.2009.01.004 Hosseini, S.E., Wahid, M.A., 2013. Biogas utilization: Experimental investigation on biogas flameless combustion in lab-scale furnace. Energy Convers. Manag. 74, 426–432. https://doi.org/10.1016/j.enconman.2013.06.026 Hwang, I.Y., Hur, D.H., Lee, J.H., Park, C.-H., Chang, I.S., Lee, J.W., Lee, E.Y., 2015. Batch conversion of methane to methanol using Methylosinus trichosporium OB3b as biocatalyst. J. Microbiol. Biotechnol. 25, 375–380. Hwang, I.Y., Lee, S.H., Choi, Y.S., Park, S.J., Na, J.G., Chang, I.S., Kim, C., Kim, H.C., Kim, Y.H., Lee, J.W., Lee, E.Y., 2014. Biocatalytic conversion of methane to methanol as a key step for development of methane-based biorefineries. J. Microbiol. Biotechnol. 24, 1597–1605. Hwang, I.Y., Nguyen, A.D., Nguyen, T.T., Nguyen, L.T., Lee, O.K., Lee, E.Y., 2018. Biological conversion of methane to chemicals and fuels: technical challenges and issues. Appl. Microbiol. Biotechnol. 102, 3071–3080. https://doi.org/10.1007/s00253-018-8842-7 Izquierdo, U., Barrio, V.L., Requies, J., Cambra, J.F., Güemez, M.B., Arias, P.L., 2013. Tri-reforming: A new biogas process for synthesis gas and hydrogen production. Int. J. Hydrog. Energy 38, 7623– 7631. https://doi.org/10.1016/j.ijhydene.2012.09.107 Jürgensen, L., Ehimen, E.A., Born, J., Holm-Nielsen, J.B., 2014. Utilization of surplus electricity from wind power for dynamic biogas upgrading: Northern Germany case study. Biomass Bioenergy 66, 126–132. https://doi.org/10.1016/j.biombioe.2014.02.032 Kalyuzhnaya, M.G., Puri, A.W., Lidstrom, M.E., 2015. Metabolic engineering in methanotrophic bacteria. Metab. Eng. 29, 142–152. https://doi.org/10.1016/j.ymben.2015.03.010 Karthikeyan, O.P., Chidambarampadmavathy, K., Cirés, S., Heimann, K., 2015. Review of Sustainable Methane Mitigation and Biopolymer Production. Crit. Rev. Environ. Sci. Technol. 45, 1579– 1610. https://doi.org/10.1080/10643389.2014.966422 Khosravi-Darani, K., Mokhtari, Z.-B., Amai, T., Tanaka, K., 2013. Microbial production of poly(hydroxybutyrate) from C1 carbon sources. Appl. Microbiol. Biotechnol. 97, 1407–1424. https://doi.org/10.1007/s00253-012-4649-0 Kits, K.D., Campbell, D.J., Rosana, A.R., Stein, L.Y., 2015. Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8. Front. Microbiol. 6. https://doi.org/10.3389/fmicb.2015.01072 Knief, C., 2015. Diversity and Habitat Preferences of Cultivated and Uncultivated Aerobic Methanotrophic Bacteria Evaluated Based on pmoA as Molecular Marker. Front. Microbiol. 6. https://doi.org/10.3389/fmicb.2015.01346 Kraft, B., Strous, M., Tegetmeyer, H.E., 2011. Microbial nitrate respiration – Genes, enzymes and environmental distribution. J. Biotechnol., New Frontiers in Microbial Genome Research 155, 104–117. https://doi.org/10.1016/j.jbiotec.2010.12.025 Krishna Reddy, Y.V., Adamala, S., Levlin, E.K., Reddy, K.S., 2017. Enhancing nitrogen removal efficiency of domestic wastewater through increased total efficiency in sewage treatment (ITEST) pilot plant in cold climatic regions of Baltic Sea. Int. J. Sustain. Built Environ. 6, 351–358. https://doi.org/10.1016/j.ijsbe.2017.05.002 LAAN, G.P.V.D., BEENACKERS, A.A.C.M., 1999. Kinetics and Selectivity of the Fischer–Tropsch Synthesis: A Literature Review. Catal. Rev. 41, 255–318. https://doi.org/10.1081/CR-100101170 Lanzini, A., Leone, P., 2010. Experimental investigation of direct internal reforming of biogas in solid oxide fuel cells. Int. J. Hydrog. Energy 35, 2463–2476. https://doi.org/10.1016/j.ijhydene.2009.12.146 Lee, H.-J., Bae, J.-H., Cho, K.-M., 2001. Simultaneous nitrification and denitrification in a mixed methanotrophic culture. Biotechnol. Lett. 23, 935–941. https://doi.org/10.1023/A:1010566616907 69 Lee, S.G., Goo, J.H., Kim, H.G., Oh, J.-I., Kim, Y.M., Kim, S.W., 2004. Optimization of methanol biosynthesis from methane using Methylosinus trichosporium OB3b. Biotechnol. Lett. 26, 947– 950. https://doi.org/10.1023/B:bile.0000025908.19252.63 Liu, H., Hu, Z., Zhang, Y., Zhang, J., Xie, H., Liang, S., 2018. Microbial nitrogen removal of ammonia wastewater in poly (butylenes succinate)-based constructed wetland: effect of dissolved oxygen. Appl. Microbiol. Biotechnol. 102, 9389–9398. https://doi.org/10.1007/s00253-018-9386-6 Lunsford, J.H., 2000. Catalytic conversion of methane to more useful chemicals and fuels: a challenge for the 21st century. Catal. Today 63, 165–174. https://doi.org/10.1016/S0920-5861(00)00456-9 Mamais, D., Noutsopoulos, C., Dimopoulou, A., Stasinakis, A., Lekkas, T.D., 2015. Wastewater treatment process impact on energy savings and greenhouse gas emissions. Water Sci. Technol. J. Int. Assoc. Water Pollut. Res. 71, 303–308. https://doi.org/10.2166/wst.2014.521 Massara, T.M., Malamis, S., Guisasola, A., Baeza, J.A., Noutsopoulos, C., Katsou, E., 2017. A review on nitrous oxide (N2O) emissions during biological nutrient removal from municipal wastewater and sludge reject water. Sci. Total Environ. 596–597, 106–123. https://doi.org/10.1016/j.scitotenv.2017.03.191 Milewski, J., Discepoli, G., Desideri, U., 2014. Modeling the performance of MCFC for various fuel and oxidant compositions. Int. J. Hydrog. Energy 39, 11713–11721. https://doi.org/10.1016/j.ijhydene.2014.05.151 Modin, O., Fukushi, K., Yamamoto, K., 2007. Denitrification with methane as external carbon source. Water Res. 41, 2726–2738. https://doi.org/10.1016/j.watres.2007.02.053 Monteith, H.D., Sahely, H.R., MacLean, H.L., Bagley, D.M., 2005. A Rational Procedure for Estimation of Greenhouse-Gas Emissions from Municipal Wastewater Treatment Plants. Water Environ. Res. 77, 390–403. https://doi.org/10.1002/j.1554-7531.2005.tb00298.x Nyerges, G., Stein, L.Y., 2009. Ammonia cometabolism and product inhibition vary considerably among species of methanotrophic bacteria. FEMS Microbiol. Lett. 297, 131–136. https://doi.org/10.1111/j.1574-6968.2009.01674.x Obaja, D., Macé, S., Costa, J., Sans, C., Mata-Alvarez, J., 2003. Nitrification, denitrification and biological phosphorus removal in piggery wastewater using a sequencing batch reactor. Bioresour. Technol. 87, 103–111. https://doi.org/10.1016/S0960-8524(02)00229-8 Olah, G.A., Goeppert, A., Czaun, M., Prakash, G.K.S., 2013. Bi-reforming of Methane from Any Source with Steam and Carbon Dioxide Exclusively to Metgas (CO–2H2) for Methanol and Hydrocarbon Synthesis. J. Am. Chem. Soc. 135, 648–650. https://doi.org/10.1021/ja311796n Park, D., Lee, J., 2013. Biological conversion of methane to methanol. Korean J. Chem. Eng. 30, 977– 987. https://doi.org/10.1007/s11814-013-0060-5 Park, S., Hanna, L., Taylor, R.T., Droege, M.W., 1991. Batch cultivation of Methylosinus trichosporium OB3b. I: Production of soluble methane monooxygenase [WWW Document]. Biotechnol. Bioeng. https://doi.org/10.1002/bit.260380412 Patel, S.K.S., Mardina, P., Kim, D., Kim, S.-Y., Kalia, V.C., Kim, I.-W., Lee, J.-K., 2016a. Improvement in methanol production by regulating the composition of synthetic gas mixture and raw biogas. Bioresour. Technol. 218, 202–208. https://doi.org/10.1016/j.biortech.2016.06.065 Patel, S.K.S., Selvaraj, C., Mardina, P., Jeong, J.-H., Kalia, V.C., Kang, Y.C., Lee, J.-K., 2016b. Enhancement of methanol production from synthetic gas mixture by Methylosinus sporium through covalent immobilization. Appl. Energy 171, 383–391. https://doi.org/10.1016/j.apenergy.2016.03.022 Periana, null, Taube, null, Gamble, null, Taube, null, Satoh, null, Fujii, null, 1998. Platinum catalysts for the high-yield oxidation of methane to a methanol derivative. Science 280, 560–564. https://doi.org/10.1126/science.280.5363.560 Perry Murray, E., Tsai, T., Barnett, S.A., 1999. A direct-methane fuel cell with a ceria-based anode. Nature 400, 649–651. https://doi.org/10.1038/23220 70 Pieja, A.J., Rostkowski, K.H., Criddle, C.S., 2011. Distribution and selection of poly-3-hydroxybutyrate production capacity in methanotrophic proteobacteria. Microb. Ecol. 62, 564–573. https://doi.org/10.1007/s00248-011-9873-0 Pöschl, M., Ward, S., Owende, P., 2010. Evaluation of energy efficiency of various biogas production and utilization pathways. Appl. Energy 87, 3305–3321. https://doi.org/10.1016/j.apenergy.2010.05.011 Praetorius, B., Bauknecht, D., Cames, M., Fischer, C., Pehnt, M., Schumacher, K., Voß, J.-P. (Eds.), 2009. Micro Cogeneration, in: Innovation for Sustainable Electricity Systems: Exploring the Dynamics of Energy Transitions, Sustainability and Innovation. Physica-Verlag HD, Heidelberg, pp. 45–75. https://doi.org/10.1007/978-3-7908-2076-8_4 Purwono, Arya Rezagama, n.d. Ammonia-Nitrogen (NH3-N) and Ammonium-Nitrogen (NH4+-N) Equilibrium on The Process of Removing Nitrogen By Using Tubular Plastic Media [WWW Document]. URL https://www.jmaterenvironsci.com/Document/vol8/vol8_NS/522-JMES-2876Purwono.pdf (accessed 3.19.19). Robert Smith, 1978. Total Energy Consumption for Municipal Wastewater Treatment [WWW Document]. URL https://nepis.epa.gov/Exe/ZyNET.exe/9100SR0P.TXT?ZyActionD=ZyDocument&Client=EPA& Index=1976+Thru+1980&Docs=&Query=&Time=&EndTime=&SearchMethod=1&TocRestrict =n&Toc=&TocEntry=&QField=&QFieldYear=&QFieldMonth=&QFieldDay=&IntQFieldOp=0 &ExtQFieldOp=0&XmlQuery=&File=D%3A%5Czyfiles%5CIndex%20Data%5C76thru80%5C Txt%5C00000018%5C9100SR0P.txt&User=ANONYMOUS&Password=anonymous&SortMeth od=h%7C&MaximumDocuments=1&FuzzyDegree=0&ImageQuality=r75g8/r75g8/x150y150g16/i425&Di splay=hpfr&DefSeekPage=x&SearchBack=ZyActionL&Back=ZyActionS&BackDesc=Results% 20page&MaximumPages=1&ZyEntry=1&SeekPage=x&ZyPURL# (accessed 5.1.19). Rostkowski, K.H., Criddle, C.S., Lepech, M.D., 2012. Cradle-to-Gate Life Cycle Assessment for a Cradle-to-Cradle Cycle: Biogas-to-Bioplastic (and Back). Environ. Sci. Technol. 46, 9822–9829. https://doi.org/10.1021/es204541w Rostkowski, K.H., Pfluger, A.R., Criddle, C.S., 2013. Stoichiometry and kinetics of the PHB-producing Type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP. Bioresour. Technol. 132, 71–77. https://doi.org/10.1016/j.biortech.2012.12.129 Saxena, S., Bedoya, I.D., 2013. Fundamental phenomena affecting low temperature combustion and HCCI engines, high load limits and strategies for extending these limits. Prog. Energy Combust. Sci. 39, 457–488. https://doi.org/10.1016/j.pecs.2013.05.002 Schmersahl, R., Mumme, J., Scholz, V., 2007. Farm-Based Biogas Production, Processing, and Use in Polymer Electrolyte Membrane (PEM) Fuel Cells. Ind. Eng. Chem. Res. 46, 8946–8950. https://doi.org/10.1021/ie071292g Semrau, J.D., DiSpirito, A.A., Vuilleumier, S., 2011. Facultative methanotrophy: false leads, true results, and suggestions for future research. FEMS Microbiol. Lett. 323, 1–12. https://doi.org/10.1111/j.1574-6968.2011.02315.x Semrau, J.D., DiSpirito, A.A., Yoon, S., 2010. Methanotrophs and copper. FEMS Microbiol. Rev. 34, 496–531. https://doi.org/10.1111/j.1574-6976.2010.00212.x Sheets, J.P., Ge, X., Li, Y.-F., Yu, Z., Li, Y., 2016. Biological conversion of biogas to methanol using methanotrophs isolated from solid-state anaerobic digestate. Bioresour. Technol. 201, 50–57. https://doi.org/10.1016/j.biortech.2015.11.035 Sheets, J.P., Lawson, K., Ge, X., Wang, L., Yu, Z., Li, Y., 2017. Development and evaluation of a trickle bed bioreactor for enhanced mass transfer and methanol production from biogas. Biochem. Eng. J. 122, 103–114. https://doi.org/10.1016/j.bej.2017.03.006 Shiratori, Y., Ijichi, T., Oshima, T., Sasaki, K., 2010. Internal reforming SOFC running on biogas. Int. J. Hydrog. Energy, The 10th Chinese Hydrogen Energy Conference 35, 7905–7912. https://doi.org/10.1016/j.ijhydene.2010.05.064 71 Shiratori, Y., Oshima, T., Sasaki, K., 2008. Feasibility of direct-biogas SOFC. Int. J. Hydrog. Energy 33, 6316–6321. https://doi.org/10.1016/j.ijhydene.2008.07.101 Smith, T.J., Trotsenko, Y.A., Murrell, J.C., 2010. Physiology and Biochemistry of the Aerobic Methane Oxidizing Bacteria, in: Timmis, K.N. (Ed.), Handbook of Hydrocarbon and Lipid Microbiology. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 765–779. https://doi.org/10.1007/978-3-54077587-4_58 Stein, L.Y., Klotz, M.G., 2011. Nitrifying and denitrifying pathways of methanotrophic bacteria. Biochem. Soc. Trans. 39, 1826–1831. https://doi.org/10.1042/BST20110712 Stocker, T.F., Qin, D., Plattner, G.-K., Tignor, M.M.B., Allen, S.K., Boschung, J., Nauels, A., Xia, Y., Bex, V., Midgley, P.M., n.d. Working Group I Contribution to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change 14. Strong, P.J., Xie, S., Clarke, W.P., 2015. Methane as a Resource: Can the Methanotrophs Add Value? Environ. Sci. Technol. 49, 4001–4018. https://doi.org/10.1021/es504242n Sundstrom, E.R., Criddle, C.S., 2015. Optimization of Methanotrophic Growth and Production of Poly(3Hydroxybutyrate) in a High-Throughput Microbioreactor System. Appl. Environ. Microbiol. 81, 4767–4773. https://doi.org/10.1128/AEM.00025-15 Tang, P., Zhu, Q., Wu, Z., Ma, D., 2014. Methane activation: the past and future. Energy Environ. Sci. 7, 2580–2591. https://doi.org/10.1039/C4EE00604F Tchobanoglous, G., Burton, F.L., Metcalf & Eddy, 1991. Wastewater engineering: treatment, disposal, and reuse. McGraw-Hill, New York. US EPA, O., 2016. Global Anthropogenic Non-CO2 Greenhouse Gas Emissions: 1990-2030 [WWW Document]. US EPA. URL https://www.epa.gov/global-mitigation-non-co2-greenhousegases/global-anthropogenic-non-co2-greenhouse-gas-emissions (accessed 3.19.19). van der Ha, D., Hoefman, S., Boeckx, P., Verstraete, W., Boon, N., 2010. Copper enhances the activity and salt resistance of mixed methane-oxidizing communities. Appl. Microbiol. Biotechnol. 87, 2355–2363. http://dx.doi.org/10.1007/s00253-010-2702-4 van Teeseling, M.C.F., Pol, A., Harhangi, H.R., van der Zwart, S., Jetten, M.S.M., Op den Camp, H.J.M., van Niftrik, L., 2014. Expanding the Verrucomicrobial Methanotrophic World: Description of Three Novel Species of Methylacidimicrobium gen. nov. Appl. Environ. Microbiol. 80, 6782– 6791. https://doi.org/10.1128/AEM.01838-14 Wang, L., Tao, L., Xie, M., Xu, G., Huang, J., Xu, Y., 1993. Dehydrogenation and aromatization of methane under non-oxidizing conditions. Catal. Lett. 21, 35–41. https://doi.org/10.1007/BF00767368 Wang, V.C.-C., Maji, S., Chen, P.P.-Y., Lee, H.K., Yu, S.S.-F., Chan, S.I., 2017. Alkane Oxidation: Methane Monooxygenases, Related Enzymes, and Their Biomimetics. Chem. Rev. 117, 8574– 8621. https://doi.org/10.1021/acs.chemrev.6b00624 Ward, M.H., Jones, R.R., Brender, J.D., de Kok, T.M., Weyer, P.J., Nolan, B.T., Villanueva, C.M., van Breda, S.G., 2018. Drinking Water Nitrate and Human Health: An Updated Review. Int. J. Environ. Res. Public. Health 15. https://doi.org/10.3390/ijerph15071557 Wastewater Treatment Plants & Reports, 2017. . City Tor. URL https://www.toronto.ca/servicespayments/water-environment/managing-sewage-in-toronto/wastewater-treatment-plants-andreports/ (accessed 3.19.19). Wendlandt, K.-D., Geyer, W., Mirschel, G., Al-Haj Hemidi, F., 2005. Possibilities for controlling a PHB accumulation process using various analytical methods. J. Biotechnol. 117, 119–129. https://doi.org/10.1016/j.jbiotec.2005.01.007 Wendlandt, K.D., Jechorek, M., Helm, J., Stottmeister, U., 2001. Producing poly-3-hydroxybutyrate with a high molecular mass from methane. J. Biotechnol. 86, 127–133. Whittenbury, R., Phillips, K.C., Wilkinson, J.F., 1970. Enrichment, Isolation and Some Properties of Methane-utilizing Bacteria. Microbiology 61, 205–218. https://doi.org/10.1099/00221287-61-2205 72 Wongchanapai, S., Iwai, H., Saito, M., Yoshida, H., 2013. Performance evaluation of a direct-biogas solid oxide fuel cell-micro gas turbine (SOFC-MGT) hybrid combined heat and power (CHP) system. J. Power Sources 223, 9–17. https://doi.org/10.1016/j.jpowsour.2012.09.037 Xu, J., Zhou, W., Li, Z., Wang, J., Ma, J., 2009. Biogas reforming for hydrogen production over nickel and cobalt bimetallic catalysts. Int. J. Hydrog. Energy, 4th Dubrovnik Conference 34, 6646–6654. https://doi.org/10.1016/j.ijhydene.2009.06.038 Yang, Y.-C., Lee, B.-J., Chun, Y.-N., 2009. Characteristics of methane reforming using gliding arc reactor. Energy 34, 172–177. https://doi.org/10.1016/j.energy.2008.11.006 Yoo, Y.-S., Han, J.-S., Ahn, C.-M., Kim, C.-G., 2015. Comparative enzyme inhibitive methanol production by Methylosinus sporium from simulated biogas. Environ. Technol. 36, 983–991. https://doi.org/10.1080/09593330.2014.971059 Young, C.H., Hanrahan, M.B., 2009. SS: CNG Transportation Technology in 2009, Marine CNG - Why hasn&apos;t it happened? Presented at the Offshore Technology Conference, Offshore Technology Conference. https://doi.org/10.4043/20145-MS Yver-Kwok, C.E., Müller, D., Caldow, C., Lebegue, B., Mønster, J.G., Rella, C.W., Scheutz, C., Schmidt, M., Ramonet, M., Warneke, T., Broquet, G., Ciais, P., 2013. Estimation of waste water treatment plant methane emissions: methodology and results from a short campaign. Atmospheric Meas. Tech. Discuss. 6, 9181–9224. https://doi.org/10.5194/amtd-6-9181-2013 Zhang, T., Zhou, J., Wang, X., Zhang, Y., 2017. Coupled effects of methane monooxygenase and nitrogen source on growth and poly-β-hydroxybutyrate (PHB) production of Methylosinus trichosporium OB3b. J. Environ. Sci. China 52, 49–57. https://doi.org/10.1016/j.jes.2016.03.001 Zhang, W., Ge, X., Li, Y.-F., Yu, Z., Li, Y., 2016. Isolation of a methanotroph from a hydrogen sulfiderich anaerobic digester for methanol production from biogas. Process Biochem. 51, 838–844. https://doi.org/10.1016/j.procbio.2016.04.003 Zhu, J., Xu, X., Yuan, M., Wu, H., Ma, Z., Wu, W., 2017. Optimum O2:CH4 Ratio Promotes the Synergy between Aerobic Methanotrophs and Denitrifiers to Enhance Nitrogen Removal. Front. Microbiol. 8, 1112. https://doi.org/10.3389/fmicb.2017.01112 73 APPENDIX A Data analysis of Growth Rate, Growth Yield, Methane Uptake Rate, Methane and Oxygen Consumption: Day sample Cycle # 23-Oct 1 24-Oct 2 25-Oct 3 26-Oct 4 27-Oct 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 OD OD (in) (out) 2.185 2.11 2.08 1.99 2.07 2.17 2.11 2.13 2.11 2.19 2.02 2.01 2.06 2.03 2.04 2.05 1.92 1.84 2 1.83 2 2.04 2.27 2.12 2.11 2.03 2.1 2.19 2.14 1.96 1.75 1.75 1.82 2.26 2.36 2.56 2.39 2.34 2.47 2.21 2.32 2.21 2.36 2.59 2.62 1.81 1.91 1.92 2.48 2.56 2.56 2.2 2.25 2.19 2.16 2.2 2.11 2.48 2.47 2.55 1.76 1.17 1.8 2.5 2.11 2.08 2.08 2.77 2.6 2.76 3.01 3.08 2.94 3.21 3.06 3.03 2.99 3.08 2.97 2.14 2 2.05 2.67 2.73 2.84 3.03 3.27 3.22 2.88 2.89 2.85 3.23 3.44 3.36 2 1.99 2.12 3.03 3.2 3.17 3.34 3.25 3.35 3 3.13 3.1 3.26 3.39 3.44 2.02 1.27 2 3.2 3.25 3.18 3.08 3.15 3.03 2.95 2.93 2.9 3.38 3.18 3.45 1.15 0.96 1.19 2.73 TSS Growth Growth (mg/L) time(hr) inc -35.714 -14.286 0.000 371.429 252.381 280.952 428.571 452.381 395.238 485.714 495.238 485.714 442.857 500.000 442.857 42.857 38.095 100.000 319.048 428.571 400.000 471.429 476.190 523.810 366.667 409.524 357.143 495.238 619.048 666.667 119.048 114.286 142.857 366.667 400.000 290.476 452.381 433.333 419.048 376.190 385.714 423.810 428.571 380.952 390.476 100.000 -304.762 38.095 342.857 328.571 295.238 419.048 428.571 400.000 376.190 347.619 376.190 428.571 338.095 428.571 -290.476 -100.000 -290.476 109.524 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 23 23 23 23 23 23 23 23 23 22.5 22.5 22.5 22.5 22.5 22.5 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 23 23 23 23 CO2 Gas rate rate volume Initial initial initial initial volume (day-1) -0.70 -0.29 0.00 6.55 4.54 4.79 7.03 7.29 6.57 7.56 8.19 8.10 7.37 8.22 7.43 1.09 1.03 2.73 7.25 9.97 8.77 9.87 9.12 10.41 7.80 8.83 7.66 9.70 11.77 13.30 2.78 2.68 3.18 6.08 6.31 4.44 6.92 6.79 6.31 6.47 6.34 7.15 6.83 5.71 5.77 2.63 -9.66 0.98 6.08 5.70 5.18 8.00 8.00 7.72 7.42 6.83 7.57 7.37 6.03 7.20 -8.75 -4.12 -8.52 1.84 (day-1) -0.035 -0.01 0.00 0.33 0.23 0.24 0.35 0.36 0.33 0.38 0.41 0.40 0.37 0.41 0.37 0.05 0.05 0.14 0.36 0.50 0.44 0.49 0.46 0.52 0.39 0.44 0.38 0.48 0.59 0.66 0.14 0.13 0.16 0.30 0.32 0.22 0.35 0.34 0.32 0.32 0.32 0.36 0.34 0.29 0.29 0.13 -0.48 0.05 0.30 0.29 0.26 0.40 0.40 0.39 0.37 0.34 0.38 0.37 0.30 0.36 -0.44 -0.21 -0.43 0.09 (ml) 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 Gas (mg/l) 528.2 549.6 532.2 552.2 529.1 310.8 538.4 556.0 543.9 532.1 533.5 547.6 536.1 557.0 486.8 595.6 576.8 558.3 567.1 576.5 547.8 568.9 592.4 580.2 575.8 572.6 540.7 574.5 561.3 540.6 544.1 534.7 502.7 504.3 509.4 460.6 474.4 459.5 478.6 475.2 473.5 527.6 507.8 489.2 562.0 559.0 374.5 528.0 496.3 526.2 511.4 721.6 296.7 495.4 497.9 627.1 476.0 517.0 490.1 481.4 598.1 399.3 549.4 570.6 O2 (mg/l) 177.0 162.3 235.1 265.6 253.7 139.4 266.6 264.3 212.9 251.9 259.3 261.7 229.0 243.7 402.2 208.9 245.4 290.4 209.6 223.3 308.6 222.3 194.3 197.3 202.6 236.0 308.7 216.9 253.0 296.7 330.1 347.1 387.9 302.7 258.2 308.7 377.7 366.2 319.1 323.3 337.5 328.7 330.8 219.7 187.0 400.3 332.2 445.8 328.2 313.0 290.1 306.3 198.2 348.0 262.4 341.2 336.3 277.2 327.6 356.9 391.3 375.8 467.9 202.4 N2 (mg/l) 0.0 0.0 0.0 240.3 239.3 142.1 253.4 246.8 284.6 267.4 262.0 246.4 278.6 261.8 202.8 0.0 0.0 0.0 266.4 260.7 228.6 262.3 277.5 277.3 277.3 265.9 239.4 275.7 262.3 247.6 0.0 0.0 0.0 247.2 278.4 261.7 229.5 236.6 259.1 259.9 254.9 236.4 236.7 247.4 295.2 0.0 0.0 0.0 243.4 234.3 256.9 126.5 141.1 228.3 281.9 166.6 244.2 259.1 239.3 230.3 0.0 0.0 0.0 272.7 CH4 (mg/l) 71.2 50.3 48.1 44.9 55.2 77.9 48.1 53.0 57.4 51.2 53.4 46.3 0.0 61.0 0.0 61.9 0.0 0.0 56.1 0.0 0.0 52.5 0.0 49.8 0.0 48.5 0.0 0.0 0.0 0.0 54.1 46.3 0.0 0.0 46.7 65.9 0.0 0.0 0.0 45.0 0.0 52.8 0.0 0.0 46.3 49.0 46.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 46.3 0.0 0.0 53.0 (ml) 373.7 391.9 1028.5 258 160 118 207 1701 190 206 215 215 198 214 251 396.5 405.8 397.1 277 508 259 206 190 177 219 174 238 269 213 250 418.3 405.9 402.5 157 218 191 242 277 236 253 253 244 207 210 178 411.5 291.2 396.5 441 768 234 263 158 297 392 316 226 212 166 264 417.3 370.3 394.5 217 O2 final N2 final (mg/l) (mg/l) 522.7 528.9 145.5 300.7 170.3 88.9 71.2 0.0 90.1 96.1 103.7 107.3 76.1 75.9 90.3 541.4 532.8 519.1 321.6 120.4 262.8 162.5 178.1 186.6 228.2 155.9 156.6 65.5 112.7 109.9 505.0 496.5 469.4 94.5 95.9 81.8 82.0 57.2 79.5 116.8 111.7 158.2 71.3 63.0 70.8 500.2 454.5 471.7 62.0 35.0 101.8 63.7 107.8 74.9 87.6 237.5 121.1 92.9 91.0 79.7 523.6 504.5 461.8 165.7 189.4 165.6 91.4 411.7 635.0 473.2 514.9 62.2 447.4 490.1 483.3 486.1 463.7 455.4 641.9 210.7 241.9 292.5 302.3 175.7 475.9 431.1 409.8 446.2 369.5 543.5 518.8 322.1 474.2 474.2 315.7 342.0 385.6 771.0 473.8 648.1 625.2 528.6 540.3 510.7 533.5 540.0 640.2 418.9 420.0 389.1 456.4 449.7 297.4 163.1 495.0 466.8 502.0 468.7 267.5 431.9 595.8 523.9 787.8 541.7 375.1 405.9 474.5 373.1 74 CH4 CO2 final final (mg/l) 0.0 0.0 0.0 185.3 96.1 159.7 176.3 12.8 206.3 177.0 173.5 119.3 211.8 188.0 123.4 0.0 0.0 0.0 240.4 74.8 170.0 192.2 186.6 195.7 221.6 126.8 131.0 120.9 173.7 168.2 0.0 0.0 0.0 66.4 217.6 145.5 154.8 152.1 204.2 215.7 206.3 170.7 120.9 173.7 190.0 0.0 0.0 0.0 73.6 81.4 150.4 23.1 162.2 58.6 129.1 39.5 75.6 179.0 70.6 124.6 0.0 0.0 0.0 201.8 (mg/l) 62.75 61.86 81.44 261.2 294.6 435.2 485.5 445.0 398.7 381.8 355.1 382.7 435.7 445.0 403.2 89.00 171.77 135.73 314.2 317.7 312.4 406.3 377.4 420.1 372.9 318.2 321.3 431.2 418.7 424.1 82.77 82.77 76.54 309.3 363.1 310.2 341.8 318.6 368.5 317.3 313.7 348.9 353.8 348.0 347.1 84.55 226.06 115.70 402.3 151.3 356.4 318.6 392.9 285.2 303.5 312.8 269.7 380.9 316.8 363.1 85.44 185.12 121.93 464.6 O2 % O2 CH4 % CH4 CO2 consum Consum consum Consum Produce ed (mg) 15.95 12.58 63.26 143.31 184.41 113.85 200.64 222.38 200.42 193.07 191.13 195.96 199.42 206.55 172.07 23.54 14.55 17.14 137.64 169.41 150.95 194.03 203.19 199.09 180.27 201.95 179.01 212.18 200.45 188.72 6.42 12.37 12.15 186.87 182.85 168.64 169.94 167.95 172.67 160.52 161.13 172.52 188.38 182.47 212.18 17.75 17.47 24.16 171.18 183.58 180.69 271.92 101.64 175.93 164.79 175.79 163.05 187.14 180.91 171.59 20.74 -27.09 37.61 192.30 ed 7.5 5.7 29.7 64.9 87.1 91.6 93.2 100.0 92.1 90.7 89.6 89.5 93.0 92.7 88.4 9.9 6.3 7.7 60.7 73.5 68.9 85.3 85.7 85.8 78.3 88.2 82.8 92.3 89.3 87.3 3.0 5.8 6.0 92.6 89.7 91.5 89.6 91.4 90.2 84.4 85.1 81.7 92.7 93.2 94.4 7.9 11.7 11.4 86.2 87.2 88.3 94.2 85.7 88.8 82.7 70.1 85.6 90.5 92.3 89.1 8.7 -17.0 17.1 84.2 ed (mg) 0.00 0.00 0.00 48.28 80.36 38.02 64.84 76.91 74.57 70.59 67.58 72.88 69.61 64.48 50.20 0.00 0.00 0.00 39.89 66.26 47.35 65.27 75.61 76.31 62.31 84.33 64.58 77.71 67.86 56.95 0.00 0.00 0.00 88.45 63.90 76.96 54.41 52.47 55.40 49.37 49.74 52.97 69.70 62.53 84.22 0.00 0.00 0.00 64.86 31.20 67.50 44.56 30.82 73.90 62.10 54.16 80.59 65.74 83.98 59.27 0.00 0.00 0.00 65.27 ed #DIV/0! #DIV/0! #DIV/0! 50.2 84.0 66.9 64.0 77.9 65.5 66.0 64.5 73.9 62.5 61.6 61.9 #DIV/0! #DIV/0! #DIV/0! 37.4 63.5 51.8 62.2 68.1 68.8 56.2 79.3 67.4 70.5 64.7 57.5 #DIV/0! #DIV/0! #DIV/0! 89.5 57.4 73.5 59.3 55.5 53.5 47.5 48.8 56.0 73.6 63.2 71.3 #DIV/0! #DIV/0! #DIV/0! 66.6 33.3 65.7 88.0 54.6 80.9 55.1 81.3 82.5 63.4 87.7 64.3 #DIV/0! #DIV/0! #DIV/0! 59.8 d (mg) -5.03 4.13 64.53 49.4 25.0 20.1 81.3 735.7 52.9 58.0 54.8 63.9 86.1 70.9 101.1 10.55 69.70 53.90 64.7 161.5 81.0 62.8 71.6 54.4 81.8 35.9 76.5 116.1 89.4 106.1 12.99 15.08 30.80 48.6 60.5 32.8 82.6 88.3 87.0 62.3 79.4 63.8 73.1 73.0 43.3 15.21 47.13 45.88 177.6 116.2 83.5 83.6 62.1 84.7 119.0 98.8 60.9 80.6 52.7 95.7 17.14 68.56 48.10 79.6 O2 CH4 (mg/hr) (mg/hr) 0.66 0.52 2.64 5.97 7.68 4.74 8.36 9.27 8.35 8.04 7.96 8.16 8.31 8.61 7.17 1.24 0.77 0.90 7.24 8.92 7.94 10.21 10.69 10.48 9.49 10.63 9.42 11.17 10.55 9.93 0.28 0.54 0.53 8.12 7.95 7.33 7.39 7.30 7.51 7.13 7.16 7.67 8.37 8.11 9.43 0.89 0.87 1.21 8.56 9.18 9.03 13.60 5.08 8.80 8.24 8.79 8.15 9.36 9.05 8.58 0.90 -1.18 1.64 8.36 0.00 0.00 0.00 2.01 3.35 1.58 2.70 3.20 3.11 2.94 2.82 3.04 2.90 2.69 2.09 0.00 0.00 0.00 2.10 3.49 2.49 3.44 3.98 4.02 3.28 4.44 3.40 4.09 3.57 3.00 0.00 0.00 0.00 3.85 2.78 3.35 2.37 2.28 2.41 2.19 2.21 2.35 3.10 2.78 3.74 0.00 0.00 0.00 3.24 1.56 3.38 2.23 1.54 3.70 3.11 2.71 4.03 3.29 4.20 2.96 0.00 0.00 0.00 2.84 obs Yield #DIV/0! #DIV/0! #DIV/0! 7.69 3.14 7.39 6.61 5.88 5.30 6.88 7.33 6.66 6.36 7.75 8.82 #DIV/0! #DIV/0! #DIV/0! 8.00 6.47 8.45 7.22 6.30 6.86 5.88 4.86 5.53 6.37 9.12 11.71 #DIV/0! #DIV/0! #DIV/0! 4.15 6.26 3.77 8.31 8.26 7.56 7.62 7.75 8.00 6.15 6.09 4.64 #DIV/0! #DIV/0! #DIV/0! 5.29 10.53 4.37 9.40 13.91 5.41 6.06 6.42 4.67 6.52 4.03 7.23 #DIV/0! #DIV/0! #DIV/0! 1.68 Cycle # Day 5 28-Oct 6 29-Oct 7 30-Oct 8 01-Nov 10 TSS OD OD (in) (out) 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 2.55 2.36 2.22 1.93 1.93 1.83 2.1 2.03 2.02 1.99 1.15 0.95 1.05 2.26 2.2 2.35 2.26 2.31 2.37 2.32 2.35 2.16 2.39 2.41 2.26 0.88 0.8 0.9 1.98 1.98 2.06 1.97 2.36 2.33 2.19 2.08 2.15 2.24 2.28 2.28 0.84 0.73 0.82 2.06 1.99 1.94 1.75 2.3 2.42 2.35 1.98 2.17 2.23 2.38 2.4 2.94 3.27 3.23 3.3 2.84 2.78 2.97 3.29 3.17 3.29 0.91 0.9 0.95 2.61 2.71 2.74 3.27 3.35 3.35 3.15 3.13 2.96 3.16 3.37 3.27 0.67 0.57 0.64 2.56 2.38 2.33 2.4 3.41 3.16 2.88 2.64 2.84 3.09 3.29 3.3 0.78 0.71 0.89 2.8 2.7 2.45 3 3.05 3.17 2.92 2.76 2.52 3.2 3.29 3.29 185.714 433.333 480.952 652.381 433.333 452.381 414.286 600.000 547.619 619.048 -114.286 -23.810 -47.619 166.667 242.857 185.714 480.952 495.238 466.667 395.238 371.429 380.952 366.667 457.143 480.952 -100.000 -109.524 -123.810 276.190 190.476 128.571 204.762 500.000 395.238 328.571 266.667 328.571 404.762 480.952 485.714 -28.571 -9.524 33.333 352.381 338.095 242.857 595.238 357.143 357.143 271.429 371.429 166.667 461.905 433.333 423.810 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 0.83 0.7 0.86 2.16 2.39 2.18 2.21 1.98 2.22 1.91 0.88 0.68 0.84 2.92 2.98 2.87 3.25 2.81 2.7 2.25 23.810 -9.524 -9.524 361.905 280.952 328.571 495.238 395.238 228.571 161.905 sample Growth Growth CO2 Gas rate rate volume Initial initial initial initial volume (day-1) (day-1) (ml) (mg/l) (mg/l) (mg/l) (mg/l) (ml) 23 23 23 23 23 23 23 23 23 23 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 2.97 6.75 7.74 10.93 7.96 8.60 7.16 9.89 9.25 10.28 -5.59 -1.30 -2.40 3.45 4.99 3.68 8.77 8.82 8.22 7.28 6.83 7.50 6.66 7.97 8.77 -6.34 -7.86 -7.91 5.98 4.30 2.88 4.61 8.52 7.08 6.37 5.56 6.48 7.47 8.49 8.56 -1.98 -0.74 2.18 8.12 8.07 6.20 14.04 7.48 7.16 5.77 8.78 3.98 9.53 8.56 8.34 0.15 0.34 0.39 0.55 0.40 0.43 0.36 0.49 0.46 0.51 -0.28 -0.06 -0.12 0.17 0.25 0.18 0.44 0.44 0.41 0.36 0.34 0.38 0.33 0.40 0.44 -0.32 -0.39 -0.40 0.30 0.21 0.14 0.23 0.43 0.35 0.32 0.28 0.32 0.37 0.42 0.43 -0.10 -0.04 0.11 0.41 0.40 0.31 0.70 0.37 0.36 0.29 0.44 0.20 0.48 0.43 0.42 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 519.1 575.6 551.0 535.4 559.7 556.7 536.0 555.8 580.7 373.5 619.0 611.0 582.9 565.9 561.8 552.8 575.3 565.9 539.0 561.8 562.5 533.1 539.7 557.9 535.4 577.0 579.7 557.9 584.6 575.6 554.7 585.2 580.6 560.4 571.9 567.6 543.7 541.6 581.4 563.0 632.1 627.1 605.2 494.5 557.6 501.8 523.4 495.8 507.8 526.2 521.3 497.2 515.8 505.7 489.9 275.8 212.9 231.8 292.9 218.3 220.4 288.1 217.7 160.0 225.0 429.7 453.9 490.7 223.0 239.5 287.4 212.9 245.3 328.0 230.0 245.8 328.6 292.7 238.4 298.3 516.6 529.6 558.0 205.1 222.3 273.6 196.7 217.3 265.0 203.1 227.1 300.0 276.4 194.7 231.8 506.9 239.7 553.0 271.3 229.5 313.0 243.3 298.6 312.9 226.4 254.0 283.9 251.7 261.2 327.5 249.4 269.9 268.4 243.5 274.3 267.7 243.3 273.4 297.3 160.3 0.0 0.0 0.0 262.0 260.4 238.5 269.3 256.0 218.5 262.7 255.4 220.9 234.4 256.9 230.7 0.0 0.0 0.0 269.5 263.9 244.6 274.7 266.9 246.8 278.9 265.2 233.8 247.3 277.4 265.4 0.0 0.0 0.0 271.8 267.6 234.8 275.3 246.7 239.8 277.9 265.7 257.3 268.9 267.2 235.4 82.8 44.5 49.4 0.0 0.0 63.6 52.5 0.0 0.0 0.0 50.7 62.7 0.0 0.0 0.0 45.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 23 23 23 23 23 23 23 23 23 23 1.22 -0.60 -0.49 6.24 4.59 5.70 7.95 7.23 4.07 3.41 0.06 -0.03 -0.02 0.31 0.23 0.29 0.40 0.36 0.20 0.17 400 400 400 400 400 400 400 400 400 400 369.4 405.0 521.6 509.6 449.6 475.2 516.0 297.7 478.3 508.7 433.3 238.6 309.3 282.9 376.6 295.0 244.0 194.3 347.9 278.7 0.0 0.0 0.0 238.6 220.7 254.9 260.2 399.5 219.5 246.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 (mg/L) time(hr) inc Gas O2 N2 CH4 O2 final N2 final CO2 O2 % O2 CH4 % CH4 CO2 CH4 obs ed (mg) ed ed (mg) ed d (mg) 496.2 257.7 163.8 565.0 336.3 317.9 443.8 353.4 276.5 463.8 434.3 458.1 494.8 340.6 380.8 474.2 449.1 492.4 590.7 309.5 458.2 639.4 527.2 483.8 459.1 495.7 511.0 548.5 137.0 296.9 367.5 221.6 436.4 296.1 353.9 344.5 507.6 580.9 295.4 308.0 498.1 495.0 512.4 417.5 418.7 423.8 445.2 454.6 521.9 439.9 #VALUE! 375.1 485.0 492.5 559.0 166.8 86.4 51.7 143.5 241.9 238.8 195.1 132.7 146.7 108.8 0.0 0.0 0.0 222.7 210.4 170.7 181.1 153.9 123.9 131.0 180.9 52.2 139.7 166.2 118.1 0.0 0.0 0.0 68.2 230.6 205.5 270.4 165.6 93.6 145.5 219.6 178.7 103.2 91.9 93.1 0.0 0.0 0.0 220.8 203.2 190.9 211.3 205.2 173.1 194.4 #VALUE! 224.2 198.2 199.6 161.3 460.1 416.1 398.3 391.6 304.8 322.2 302.2 388.5 417.0 405.8 66.31 81.88 85.00 311.9 311.5 388.5 410.3 410.3 413.0 356.4 395.2 293.3 369.4 426.8 449.9 66.75 93.01 168.66 342.2 245.2 242.5 174.9 400.5 429.0 386.3 294.1 367.6 375.1 430.3 431.7 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! 409.0 409.8 188.57 208.13 202.86 194.98 152.83 135.27 151.92 201.94 216.97 132.46 13.63 14.73 14.13 144.16 154.66 168.75 201.13 198.87 185.00 182.31 183.52 177.56 188.35 202.30 185.27 11.68 13.56 11.02 174.82 110.66 108.74 61.26 203.07 189.05 194.42 140.17 170.55 202.53 209.99 222.55 13.39 139.95 2.06 163.49 184.11 121.64 166.71 153.52 168.79 163.67 #VALUE! 103.58 178.93 176.29 166.32 90.8 90.4 92.0 91.0 68.3 60.8 70.9 90.8 93.4 88.7 5.5 6.0 6.1 63.7 68.8 76.3 87.4 87.9 85.8 81.1 81.6 83.3 87.3 90.7 86.5 5.1 5.9 4.9 74.8 48.1 49.0 26.2 87.4 84.3 85.0 61.7 78.4 93.5 90.3 98.8 5.3 55.8 0.9 82.6 82.6 60.6 79.6 77.4 83.1 77.8 #VALUE! 52.1 86.7 87.2 84.9 62.66 79.42 78.09 67.63 46.91 40.87 46.65 76.66 84.95 43.00 0.00 0.00 0.00 46.48 51.21 54.03 73.39 71.74 59.88 66.13 63.33 77.63 62.71 70.00 61.57 0.00 0.00 0.00 66.96 36.51 36.67 13.91 73.76 65.22 78.15 48.19 51.27 79.28 86.74 78.14 0.00 0.00 0.00 51.32 62.51 37.54 63.93 44.77 54.40 71.14 #VALUE! 35.02 66.40 64.55 56.37 62.8 73.6 72.7 69.4 42.8 38.2 47.9 70.1 71.4 67.1 #DIV/0! #DIV/0! #DIV/0! 44.4 49.2 56.6 68.1 70.1 68.5 62.9 62.0 87.9 66.9 68.1 66.7 #DIV/0! #DIV/0! #DIV/0! 62.1 34.6 37.5 12.7 69.1 66.1 70.1 45.4 54.8 80.2 78.2 73.6 #DIV/0! #DIV/0! #DIV/0! 47.2 58.4 40.0 58.1 45.4 56.7 64.0 #VALUE! 34.0 61.7 60.4 59.9 69.2 119.7 205.7 81.2 79.1 63.9 57.5 95.7 96.5 78.7 5.95 7.35 33.72 81.7 78.4 75.9 77.8 81.8 91.7 106.0 84.8 60.3 82.0 84.1 117.0 27.82 38.56 68.64 204.9 73.4 72.2 62.1 79.8 153.6 88.7 77.5 86.9 71.4 113.5 130.0 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! 86.8 96.0 8.20 9.05 8.82 8.48 6.64 5.88 6.61 8.78 9.43 5.76 0.68 0.74 0.71 7.21 7.73 8.44 10.06 9.94 9.25 9.12 9.18 8.88 9.42 10.11 9.26 0.57 0.66 0.54 8.53 5.40 5.30 2.99 9.91 9.22 9.48 6.84 8.32 9.88 10.24 10.86 0.74 7.78 0.11 9.08 10.23 6.76 9.26 8.53 9.38 9.09 #VALUE! 5.75 9.94 9.79 9.24 2.72 3.45 3.40 2.94 2.04 1.78 2.03 3.33 3.69 1.87 0.00 0.00 0.00 2.32 2.56 2.70 3.67 3.59 2.99 3.31 3.17 3.88 3.14 3.50 3.08 0.00 0.00 0.00 3.27 1.78 1.79 0.68 3.60 3.18 3.81 2.35 2.50 3.87 4.23 3.81 0.00 0.00 0.00 2.85 3.47 2.09 3.55 2.49 3.02 3.95 #VALUE! 1.95 3.69 3.59 3.13 2.96 5.46 6.16 9.65 9.24 11.07 8.88 7.83 6.45 14.40 #DIV/0! #DIV/0! #DIV/0! 3.59 4.74 3.44 6.55 6.90 7.79 5.98 5.87 4.91 5.85 6.53 7.81 #DIV/0! #DIV/0! #DIV/0! 4.12 5.22 3.51 14.72 6.78 6.06 4.20 5.53 6.41 5.11 5.55 6.22 #DIV/0! #DIV/0! #DIV/0! 6.87 5.41 6.47 9.31 7.98 6.56 3.82 #VALUE! 4.76 6.96 6.71 7.52 425.0 243.6 357.7 143.8 317.5 167.3 205.8 266.7 514.9 115.5 0.0 0.0 0.0 43.7 74.5 72.0 90.3 383.1 167.4 68.6 101.91 65.86 53.85 384.9 381.4 365.3 400.5 212.7 315.5 330.2 12.43 42.66 41.29 169.98 159.76 158.14 180.78 107.89 138.49 126.59 8.4 26.3 19.8 83.4 88.8 83.2 87.6 90.6 72.4 62.2 0.00 0.00 0.00 61.06 52.94 51.17 61.23 48.13 42.58 32.25 #DIV/0! #DIV/0! #DIV/0! 64.0 60.0 50.2 58.8 30.1 48.5 32.8 41.55 25.80 18.62 303.0 180.9 257.7 190.0 62.0 85.3 318.7 0.54 1.85 1.80 7.39 6.95 6.88 7.86 4.69 6.02 5.50 0.00 0.00 0.00 2.65 2.30 2.22 2.66 2.09 1.85 1.40 #DIV/0! #DIV/0! #DIV/0! 5.93 5.31 6.42 8.09 8.21 5.37 5.02 222 330 566 207 260 277 260 246 231 194 395.7 396.3 396.7 262 252 242 190 199 222 297 215 206 222 197 260 416.8 414.6 407.0 599 299 298 355 199 358 230 264 236 190 264 301 407.1 193.7 431.7 260 219 295 219 263 240 206 #VALUE! 303 208 212 234 85.8 66.9 31.0 92.6 273.6 315.2 240.5 82.7 66.2 87.3 591.2 579.5 552.1 313.8 278.4 215.9 152.8 137.9 137.7 142.7 193.3 173.6 123.9 105.8 111.2 525.7 526.6 521.3 98.6 399.3 380.0 486.8 146.4 98.1 149.6 329.6 198.6 74.2 85.7 8.9 588.2 572.6 556.0 132.0 177.5 267.6 195.1 170.5 143.2 227.4 #VALUE! 314.7 131.9 122.5 126.6 407.8 391.7 345.8 787 474 705 474 291 270 965 331.9 304.6 483.9 43.0 42.3 45.3 54.0 38.4 195.4 79.7 consum Consum consum Consum Produce O2 final (mg/l) (mg/l) 75 CH4 final (mg/l) (mg/l) (mg/hr) (mg/hr) Yield Cycle # Day 02-Nov 11 28-Nov 37 29-Nov 38 30-Nov 39 sample 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 OD OD (in) (out) 2.07 2.13 2.33 2.41 2.37 0.81 0.7 0.83 2.02 2.17 2.06 2.13 2.1 1.92 1.95 1.88 2.1 2.16 2.14 2.24 1.3 1.28 1.2 1.9 1.98 1.9 1.9 1.88 2.04 2.14 1.96 2.07 1.9 1.88 1.91 1.5 1.4 1.25 1.68 1.82 2.02 1.96 1.83 1.94 1.83 1.68 1.68 1.86 1.79 1.8 1.51 1.13 1.2 2.07 2.2 2.22 2.03 2.07 2.05 1.97 1.99 2 1.91 2 2 2.29 2.99 3.3 3.34 3.26 0.78 0.71 0.9 2.92 2.85 2.58 3 3.09 2.94 2.8 2.72 2.65 3.11 3.18 3.28 1.58 1.42 1.29 2.92 2.98 2.93 3.16 3.29 3.2 3.1 2.96 2.94 3.25 3.2 2.95 1.68 1.61 1.33 2.53 2.87 2.8 2.98 2.82 3.04 2.95 2.92 2.84 2.77 3.03 2.84 1.76 1.45 1.63 3.02 3.1 3.13 2.91 3.28 2.88 3.02 2.95 2.9 2.97 3.08 3.12 TSS Growth Growth (mg/L) time(hr) inc 104.762 409.524 461.905 442.857 423.810 -14.286 4.762 33.333 428.571 323.810 247.619 414.286 471.429 485.714 404.762 400.000 261.905 452.381 495.238 495.238 133.333 66.667 42.857 485.714 476.190 490.476 600.000 671.429 552.381 457.143 476.190 414.286 642.857 628.571 495.238 85.714 100.000 38.095 404.762 500.000 371.429 485.714 471.429 523.810 533.333 590.476 552.381 433.333 590.476 495.238 119.048 152.381 204.762 452.381 428.571 433.333 419.048 576.190 395.238 500.000 457.143 428.571 504.762 514.286 533.333 23 23 23 23 23 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 22 22 22 22.5 22.5 22.5 22.5 22.5 22.5 22.5 22.5 22.5 22.5 22.5 22.5 20.5 20.5 20.5 21.5 21.5 21.5 21.5 21.5 21.5 22 22 22 22 22 22 20.5 20.5 20.5 20 20 20 20 20 20 20 20 20 20 20 20 Gas O2 N2 CH4 CO2 Gas rate rate volume Initial initial initial initial volume (day-1) (day-1) (ml) (mg/l) (mg/l) (mg/l) (mg/l) (ml) 2.11 7.01 7.19 6.75 6.60 -0.95 0.36 2.04 9.21 6.84 5.66 8.57 9.64 10.60 9.04 9.23 5.85 9.11 9.88 9.52 4.24 2.26 1.58 9.03 8.60 9.10 10.62 11.64 9.45 7.82 8.67 7.41 11.18 11.09 9.13 2.65 3.27 1.45 9.02 10.00 7.23 9.22 9.51 9.86 10.22 11.76 11.20 8.58 11.23 9.78 3.58 5.81 7.12 8.96 8.15 8.16 8.55 10.86 8.08 10.10 9.33 8.82 10.43 10.20 10.50 0.11 0.35 0.36 0.34 0.33 -0.05 0.02 0.10 0.46 0.34 0.28 0.43 0.48 0.53 0.45 0.46 0.29 0.46 0.49 0.48 0.21 0.11 0.08 0.45 0.43 0.45 0.53 0.58 0.47 0.39 0.43 0.37 0.56 0.55 0.46 0.13 0.16 0.07 0.45 0.50 0.36 0.46 0.48 0.49 0.51 0.59 0.56 0.43 0.56 0.49 0.18 0.29 0.36 0.45 0.41 0.41 0.43 0.54 0.40 0.51 0.47 0.44 0.52 0.51 0.53 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 535.2 534.9 525.0 522.7 506.6 515.4 488.0 463.4 492.8 470.1 425.5 459.8 460.6 499.3 499.5 459.7 466.0 451.7 461.8 453.8 549.1 385.9 536.0 456.1 451.0 459.7 488.9 460.6 467.1 467.1 447.5 393.1 457.4 456.0 449.6 564.1 500.4 569.4 490.5 488.9 310.3 453.1 432.2 451.2 443.0 448.5 429.8 429.6 422.1 452.2 174.7 426.2 340.5 376.1 423.4 436.7 295.6 356.7 447.8 423.6 444.4 428.0 380.6 424.1 435.8 224.1 237.7 193.1 235.1 276.5 376.7 422.8 453.6 277.9 320.6 324.0 322.0 366.0 261.7 297.4 357.6 452.6 386.1 414.5 460.6 398.7 312.3 463.1 249.2 361.6 343.7 248.5 318.4 311.4 307.3 355.2 227.1 343.4 334.6 345.8 457.8 443.4 454.2 367.2 311.1 225.4 369.6 426.0 401.7 379.0 379.7 350.7 384.9 374.4 333.6 234.1 634.2 422.8 337.7 417.6 389.5 243.6 362.2 337.7 430.6 344.0 406.3 305.3 405.4 353.2 271.1 262.3 281.1 264.4 246.8 0.0 0.0 0.0 263.3 215.6 233.4 259.0 235.7 310.5 272.3 260.6 210.9 256.3 238.5 223.2 0.0 0.0 0.0 278.5 218.1 240.1 280.3 249.1 246.5 254.0 233.9 228.2 236.4 235.6 236.0 0.0 0.0 0.0 209.6 254.0 158.7 223.5 196.7 211.2 223.5 227.1 223.0 216.3 229.0 242.4 0.0 0.0 0.0 192.5 209.0 222.8 162.3 168.5 248.5 206.3 249.5 314.9 306.1 221.9 246.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 54.7 57.4 0.0 0.0 0.0 0.0 0.0 0.0 49.0 0.0 0.0 0.0 49.0 0.0 0.0 377 553 514 502 222 367.4 372.8 336.8 323 226 1158 212 241 215 266 203 209 249 181 202 409.2 813.5 #DIV/0! 588 #DIV/0! 306 648 230 666 208 #DIV/0! 1092 245 579 217 5839.3 406.0 420.6 398 778 330 #VALUE! 1698 270 405 310 245 #VALUE! 514 471 176.3 449.9 296.5 220 418 255 2210 229 503 214 258 265 250 240 232 O2 final N2 final (mg/l) (mg/l) 189.9 53.2 32.2 33.1 88.7 406.2 485.9 506.0 102.7 137.5 28.7 117.5 107.3 167.6 144.6 190.5 197.4 97.4 104.8 100.7 442.5 156.6 0.0 44.1 0.0 70.4 32.9 69.0 42.8 84.4 0.0 18.9 74.5 31.7 54.0 18.2 506.8 516.7 59.5 35.6 45.3 #VALUE! 0.0 110.8 113.5 219.1 115.1 #VALUE! 38.2 31.7 476.5 472.1 465.5 64.4 58.9 61.8 #VALUE! 76.1 47.4 112.7 124.6 173.8 21.8 39.8 36.1 237.6 172.1 150.4 187.2 498.4 410.2 453.6 538.7 344.0 566.6 111.9 606.2 607.5 486.9 447.0 705.7 866.2 619.4 917.3 912.9 389.8 153.6 0.0 169.4 0.0 449.4 153.4 554.4 186.9 591.4 0.0 83.2 559.6 231.1 638.0 31.4 436.8 431.9 369.2 160.0 273.0 #VALUE! 100.4 594.6 373.9 489.2 571.6 #VALUE! 291.3 283.5 531.2 563.9 570.4 614.3 400.0 610.5 44.1 631.8 268.8 804.6 533.7 613.1 487.6 675.5 608.9 76 CH4 CO2 final final O2 % O2 CH4 % CH4 CO2 (mg/l) (mg/l) ed (mg) ed ed (mg) ed d (mg) 151.2 72.5 59.9 69.9 190.0 0.0 0.0 0.0 118.1 223.0 16.5 245.0 178.0 304.6 181.1 269.8 173.9 275.9 248.1 190.8 0.0 0.0 0.0 79.5 0.0 160.6 57.7 186.3 72.6 56.4 0.0 14.5 128.0 50.6 100.7 0.0 0.0 0.0 110.4 61.6 86.5 #VALUE! 17.7 143.1 121.9 188.4 81.4 #VALUE! 94.5 86.3 0.0 0.0 0.0 162.3 91.8 148.0 #VALUE! 140.9 73.2 71.0 170.1 156.1 286.3 155.2 180.2 273.7 401.8 417.0 427.6 417.4 59.19 109.03 0.00 287.0 338.6 226.5 299.5 330.2 313.3 376.5 292.8 273.7 327.1 359.6 323.1 73.43 102.80 92.56 364.5 267.4 366.2 339.1 311.1 313.7 265.2 255.9 264.8 329.7 350.2 268.3 59.63 60.97 57.85 328.4 347.1 361.8 #VALUE! 266.6 275.9 282.6 197.6 222.5 #VALUE! 340.4 332.4 61.41 44.95 105.02 357.8 357.8 352.9 297.7 313.7 295.9 250.1 258.1 222.1 291.9 319.5 313.3 142.43 184.56 193.45 192.45 182.95 56.94 14.05 14.92 163.93 156.92 136.99 158.97 158.37 163.70 161.32 145.27 145.17 156.41 165.78 161.21 38.55 26.91 #DIV/0! 156.52 #DIV/0! 162.32 174.22 168.37 158.30 169.29 #DIV/0! 136.56 164.65 164.03 168.13 119.18 -5.60 10.45 172.52 167.88 109.15 #VALUE! 172.89 150.53 131.22 111.37 143.67 #VALUE! 149.21 165.98 -14.11 -41.87 -1.81 136.28 144.74 158.90 #VALUE! 125.21 155.29 145.29 145.65 125.12 146.77 160.08 165.93 66.5 86.3 92.1 92.0 90.3 27.6 7.2 8.0 83.2 83.4 80.5 86.4 86.0 82.0 80.7 79.0 77.9 86.6 89.7 88.8 17.6 17.4 #DIV/0! 85.8 #DIV/0! 88.3 89.1 91.4 84.7 90.6 #DIV/0! 86.8 90.0 89.9 93.5 52.8 -2.8 4.6 87.9 85.9 87.9 #VALUE! 100.0 83.4 74.0 62.1 83.6 #VALUE! 88.4 91.8 -20.2 -24.6 -1.3 90.6 85.5 91.0 #VALUE! 87.8 86.7 85.8 81.9 73.1 96.4 94.4 95.2 51.38 64.86 81.65 70.63 56.56 0.00 0.00 0.00 67.18 35.76 74.22 51.53 51.38 58.72 60.73 49.56 48.00 33.73 50.56 50.76 0.00 0.00 #DIV/0! 64.63 #DIV/0! 46.91 74.74 56.84 50.25 89.88 #DIV/0! 75.40 63.13 64.92 72.56 0.00 0.00 0.00 39.91 53.74 34.88 #VALUE! 48.66 45.81 40.00 32.37 69.21 #VALUE! 43.00 56.37 0.00 0.00 0.00 41.31 45.27 51.34 #VALUE! 35.11 62.60 67.34 55.92 84.59 50.72 51.50 56.87 47.4 61.8 72.6 66.8 57.3 #DIV/0! #DIV/0! #DIV/0! 63.8 41.5 79.5 49.7 54.5 47.3 55.8 47.5 56.9 32.9 53.0 56.9 #DIV/0! #DIV/0! #DIV/0! 58.0 #DIV/0! 48.9 66.7 57.1 51.0 88.5 #DIV/0! 82.6 66.8 68.9 76.9 #DIV/0! #DIV/0! #DIV/0! 47.6 52.9 55.0 #VALUE! 61.9 54.2 44.7 35.6 77.6 #VALUE! 46.9 58.1 #DIV/0! #DIV/0! #DIV/0! 53.7 54.2 57.6 #VALUE! 52.1 63.0 81.6 56.0 67.2 41.4 58.0 57.6 103.3 222.1 214.2 214.8 92.6 21.74 40.65 0.00 92.8 76.6 262.4 63.6 79.6 67.3 100.2 59.3 57.2 81.6 65.0 65.2 30.05 83.62 #DIV/0! 214.5 #DIV/0! 112.0 219.7 71.4 209.1 55.1 #DIV/0! 289.2 80.9 202.8 58.2 348.20 24.75 24.33 130.7 269.9 119.5 #VALUE! 452.5 74.6 114.6 61.3 54.6 #VALUE! 175.0 156.5 -11.07 -2.74 31.14 78.7 149.4 90.0 657.8 71.9 129.1 53.5 66.5 58.9 53.5 76.7 72.7 consum Consum consum Consum Produce O2 CH4 (mg/hr) (mg/hr) 6.19 8.02 8.41 8.37 7.95 3.00 0.74 0.79 8.63 8.26 7.21 8.37 8.34 8.62 8.49 7.65 7.64 8.23 8.73 8.48 1.75 1.22 #DIV/0! 6.96 #DIV/0! 7.21 7.74 7.48 7.04 7.52 #DIV/0! 6.07 7.32 7.29 7.47 5.81 -0.27 0.51 8.02 7.81 5.08 #VALUE! 8.04 7.00 5.96 5.06 6.53 #VALUE! 6.78 7.54 -0.69 -2.04 -0.09 6.81 7.24 7.95 #VALUE! 6.26 7.76 7.26 7.28 6.26 7.34 8.00 8.30 2.23 2.82 3.55 3.07 2.46 0.00 0.00 0.00 3.54 1.88 3.91 2.71 2.70 3.09 3.20 2.61 2.53 1.78 2.66 2.67 0.00 0.00 #DIV/0! 2.87 #DIV/0! 2.08 3.32 2.53 2.23 3.99 #DIV/0! 3.35 2.81 2.89 3.22 0.00 0.00 0.00 1.86 2.50 1.62 #VALUE! 2.26 2.13 1.82 1.47 3.15 #VALUE! 1.95 2.56 0.00 0.00 0.00 2.07 2.26 2.57 #VALUE! 1.76 3.13 3.37 2.80 4.23 2.54 2.57 2.84 obs Yield 2.04 6.31 5.66 6.27 7.49 #DIV/0! #DIV/0! #DIV/0! 6.38 9.06 3.34 8.04 9.18 8.27 6.67 8.07 5.46 13.41 9.79 9.76 #DIV/0! #DIV/0! #DIV/0! 7.52 #DIV/0! 10.46 8.03 11.81 10.99 5.09 #DIV/0! 5.49 10.18 9.68 6.83 #DIV/0! #DIV/0! #DIV/0! 10.14 9.30 10.65 #VALUE! 9.69 11.43 13.33 18.24 7.98 #VALUE! 13.73 8.79 #DIV/0! #DIV/0! #DIV/0! 10.95 9.47 8.44 #VALUE! 16.41 6.31 7.42 8.17 5.07 9.95 9.99 9.38 Cycle # Day 02-Dec 41 03-Dec 42 05-Dec New feed 44 06-Dec 45 07-Dec sample 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 OD OD (in) (out) 1.58 1.25 1.35 1.99 2.1 2.04 2.01 2 2.06 1.98 1.95 1.95 2.04 1.99 2 1.59 1.15 1.2 2.11 1.96 1.96 2.08 2.01 2.12 2.03 1.97 1.99 2.02 1.93 1.99 1.63 1.05 0.96 2.04 2 2.04 2.08 2.05 2.04 1.98 1.9 1.98 2.19 2.07 1.99 1.46 1 1 2.1 2.13 1.91 2 1.92 1.92 1.97 2.04 2 2.06 1.86 1.97 1.2 0.96 0.87 1.99 2.02 1.74 1.35 1.39 3.02 2.91 2.9 3.24 3.21 3.01 2.98 2.97 2.95 3.24 3.24 3.26 1.7 1.2 1.31 3 3.01 3.07 3 3.08 3.28 2.97 2.98 3.02 3.05 3.04 2.95 1.7 1.1 0.99 3 2.98 2.89 3.15 3.11 3.14 2.87 2.75 2.89 3.09 2.91 2.87 1.51 1.03 1.06 2.92 2.92 2.63 3.18 3.13 3.09 2.88 3.03 3.02 3.17 3.08 3.09 1.37 1.01 1.1 2.84 2.91 TSS Growth Growth (mg/L) time(hr) inc 76.190 47.619 19.048 490.476 385.714 409.524 585.714 576.190 452.381 476.190 485.714 476.190 571.429 595.238 600.000 52.381 23.810 52.381 423.810 500.000 528.571 438.095 509.524 552.381 447.619 480.952 490.476 490.476 528.571 457.143 33.333 23.810 14.286 457.143 466.667 404.762 509.524 504.762 523.810 423.810 404.762 433.333 428.571 400.000 419.048 23.810 14.286 28.571 390.476 376.190 342.857 561.905 576.190 557.143 433.333 471.429 485.714 528.571 580.952 533.333 80.952 23.810 109.524 404.762 423.810 23.25 23.25 23.25 23.25 23.25 23.25 23.5 23.5 23.5 23.75 23.75 23.75 24 24 24 21.75 21.75 21.75 22 22 22 22.25 22.25 22.25 22.5 22.5 22.5 24 24 24 21.25 21.25 21.25 21.5 21.5 21.5 21.75 21.75 21.75 22 22 22 22.25 22.25 22.25 23.5 23.5 23.5 23.5 23.5 23.5 24 24 24 23.5 23.5 23.5 24 24 24 19 19 19 19.25 19.25 CO2 Gas rate rate volume Initial initial initial initial volume (day-1) (day-1) (ml) Gas (mg/l) O2 (mg/l) N2 (mg/l) CH4 (mg/l) (ml) 1.99 1.59 0.60 8.49 6.68 7.19 9.57 9.49 7.65 8.15 8.38 8.25 9.09 9.56 9.58 1.48 0.94 1.93 7.60 9.22 9.63 7.81 9.07 9.27 8.02 8.71 8.77 8.13 8.93 7.77 0.95 1.05 0.70 8.50 8.79 7.70 9.03 9.07 9.37 8.01 7.98 8.15 7.35 7.28 7.81 0.69 0.60 1.19 6.67 6.39 6.48 9.11 9.58 9.34 7.66 7.98 8.30 8.49 9.88 8.85 3.34 1.28 5.90 8.78 9.00 0.10 0.08 0.03 0.42 0.33 0.36 0.48 0.47 0.38 0.41 0.42 0.41 0.45 0.48 0.48 0.07 0.05 0.10 0.38 0.46 0.48 0.39 0.45 0.46 0.40 0.44 0.44 0.41 0.45 0.39 0.05 0.05 0.03 0.43 0.44 0.38 0.45 0.45 0.47 0.40 0.40 0.41 0.37 0.36 0.39 0.03 0.03 0.06 0.33 0.32 0.32 0.46 0.48 0.47 0.38 0.40 0.42 0.42 0.49 0.44 0.17 0.06 0.29 0.44 0.45 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 622.7 454.7 555.8 527.6 286.9 179.5 501.3 525.9 440.9 441.6 516.7 507.3 502.5 505.7 483.9 670.1 631.4 668.0 540.9 565.9 529.6 463.9 484.8 548.9 541.4 511.9 510.6 498.6 517.4 487.8 493.3 470.1 #VALUE! 429.0 395.8 394.7 395.4 392.8 371.7 376.3 386.6 #VALUE! 389.6 #VALUE! 379.5 301.1 205.9 557.9 226.2 408.2 345.5 449.2 459.3 449.8 402.5 444.3 449.2 626.0 460.7 445.2 480.4 533.7 520.6 480.2 445.5 539.7 487.1 531.7 389.8 153.2 133.3 375.3 310.5 372.0 268.2 323.1 352.7 363.0 357.7 321.6 535.1 574.1 536.9 288.3 221.2 295.3 370.7 373.8 244.3 268.5 337.8 328.4 347.1 288.3 366.1 636.4 676.8 #VALUE! 599.3 448.6 448.7 440.2 483.1 480.8 484.0 504.8 #VALUE! 481.0 #VALUE! 511.1 400.7 264.6 665.0 162.3 316.8 219.5 421.5 424.2 401.4 325.4 447.7 426.9 418.6 361.6 422.1 712.2 695.4 704.8 533.1 433.7 0.0 0.0 0.0 186.7 132.5 74.4 208.1 232.5 192.2 202.2 225.9 215.7 209.6 211.6 203.1 0.0 0.0 0.0 238.5 268.4 234.5 223.4 209.7 252.0 249.6 220.7 227.3 215.9 240.1 209.9 0.0 0.0 0.0 81.3 198.2 198.6 204.6 182.5 200.5 197.9 171.6 #VALUE! 185.6 #VALUE! 172.0 0.0 0.0 0.0 109.4 197.6 172.5 203.7 196.5 189.8 188.2 177.7 196.7 204.2 217.4 192.0 0.0 0.0 0.0 121.8 201.2 66.8 44.9 45.4 46.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 64.5 54.7 49.4 65.9 53.4 47.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 57.0 48.5 #VALUE! 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 46.3 44.5 0.0 0.0 0.0 422.8 362.5 452.4 638 #DIV/0! 329 5206 222 287 131 324 284 288 295 285 421.4 424.9 410.6 217 687 243 723 #VALUE! 257 397 296 185 174 2196 1344 388.8 398.4 #VALUE! 341 673 2509 362 501 573 7857 8435 #VALUE! 268 #VALUE! 499 420.7 261.6 435.8 108 206 258 672 242 255 387 939 321 238 230 228 443.0 417.5 422.2 686 296 O2 final N2 final (mg/l) (mg/l) 593.0 559.1 497.4 26.7 0.0 18.4 0.0 69.6 77.2 100.9 80.9 254.5 54.7 41.6 44.4 604.3 563.9 620.4 79.7 26.7 62.1 30.4 #VALUE! 80.4 148.0 232.8 135.9 120.2 0.0 0.0 468.5 456.8 #VALUE! 50.3 28.7 0.0 56.6 50.6 37.7 0.0 0.0 #VALUE! 43.0 #VALUE! 32.9 319.0 277.9 542.7 447.1 60.5 27.8 34.7 64.3 83.5 43.7 29.4 83.5 38.8 32.7 20.9 476.1 489.2 519.8 42.8 63.7 510.6 537.5 470.1 244.4 0.0 161.8 28.8 560.1 518.6 816.8 399.4 495.9 504.1 484.8 451.8 507.9 540.5 523.0 531.4 128.8 485.9 205.0 #VALUE! 380.8 270.8 456.4 709.0 798.4 52.5 108.9 654.8 679.4 #VALUE! 703.6 266.7 71.5 486.4 385.4 335.6 24.6 23.9 #VALUE! 719.0 #VALUE! 409.4 380.9 404.6 610.4 598.5 616.1 340.6 250.7 700.1 628.7 336.1 190.8 532.1 702.4 628.0 739.8 643.0 666.3 667.7 310.9 586.2 77 CH4 CO2 O2 % O2 CH4 % CH4 CO2 final final (mg/l) (mg/l) ed (mg) consum Consum consum Consum Produce ed ed (mg) ed d (mg) 0.0 0.0 0.0 49.9 0.0 21.4 0.0 146.5 151.3 53.8 103.4 179.7 136.0 80.0 111.9 0.0 0.0 0.0 123.4 46.6 101.4 41.4 #VALUE! 115.9 107.9 157.0 83.3 29.4 9.6 12.6 0.0 0.0 0.0 108.4 51.0 9.6 77.0 63.7 66.0 0.0 10.8 #VALUE! 101.7 #VALUE! 52.3 0.0 0.0 0.0 22.7 141.4 47.8 54.2 113.1 139.7 46.1 13.9 129.2 105.5 143.7 113.7 0.0 0.0 0.0 43.4 98.0 72.98 80.99 79.66 437.4 249.6 62.7 322.6 333.3 353.3 209.2 358.7 247.4 428.1 379.1 372.5 98.35 97.01 93.45 488.2 438.8 406.3 306.2 343.1 370.2 227.4 182.9 290.6 346.2 395.6 370.2 94.34 170.88 #VALUE! 367.6 341.8 290.6 276.8 291.0 290.6 226.5 188.2 #VALUE! 299.5 #VALUE! 227.8 85.44 #VALUE! 64.53 #VALUE! 324.9 280.4 282.6 363.6 311.9 193.6 181.6 214.5 329.7 275.0 364.0 88.11 101.91 48.95 339.5 339.1 -1.63 -20.80 -2.70 194.01 #DIV/0! 65.73 200.51 194.92 154.22 163.39 180.49 130.51 185.25 190.00 180.92 13.41 12.95 12.47 199.08 207.99 196.73 163.54 #VALUE! 198.93 157.88 135.84 179.07 178.55 206.95 195.12 15.16 6.03 #VALUE! 154.49 139.02 157.88 137.66 131.72 127.08 150.52 154.63 #VALUE! 144.32 #VALUE! 135.35 -13.76 9.65 -13.33 42.00 150.81 131.04 156.36 168.15 158.57 144.07 150.13 152.88 241.18 176.75 173.30 -18.78 9.22 -11.27 162.70 159.34 -0.7 -11.4 -1.2 91.9 #DIV/0! 91.6 100.0 92.7 87.4 92.5 87.3 64.3 92.2 93.9 93.5 5.0 5.1 4.7 92.0 91.9 92.9 88.1 #VALUE! 90.6 72.9 66.3 87.7 89.5 100.0 100.0 7.7 3.2 #VALUE! 90.0 87.8 100.0 87.0 83.8 85.5 100.0 100.0 #VALUE! 92.6 #VALUE! 89.2 -11.4 11.7 -6.0 46.4 92.4 94.8 87.0 91.5 88.1 89.5 84.5 85.1 96.3 95.9 97.3 -9.8 4.3 -5.4 84.7 89.4 0.00 0.00 0.00 42.86 #DIV/0! 22.71 83.26 60.50 33.45 73.80 56.92 35.17 44.64 61.02 49.38 0.00 0.00 0.00 68.64 75.37 69.15 59.41 #VALUE! 71.07 57.02 41.78 75.50 81.27 74.95 66.95 0.00 0.00 #VALUE! -4.40 44.95 55.39 53.96 41.04 42.38 79.15 -22.72 #VALUE! 47.05 #VALUE! 42.67 0.00 0.00 0.00 41.31 49.97 56.69 44.99 51.16 40.24 57.44 58.04 37.24 56.50 53.84 50.84 0.00 0.00 0.00 18.94 51.47 #DIV/0! #DIV/0! #DIV/0! 57.4 #DIV/0! 76.3 100.0 65.1 43.5 91.3 63.0 40.8 53.3 72.1 60.8 #DIV/0! #DIV/0! #DIV/0! 71.9 70.2 73.7 66.5 #VALUE! 70.5 57.1 47.3 83.0 94.1 78.1 79.8 #DIV/0! #DIV/0! #VALUE! -13.5 56.7 69.7 65.9 56.2 52.8 100.0 -33.1 #VALUE! 63.4 #VALUE! 62.0 #DIV/0! #DIV/0! #DIV/0! 94.4 63.2 82.1 55.2 65.1 53.0 76.3 81.7 47.3 69.2 61.9 66.2 #DIV/0! #DIV/0! #DIV/0! 38.9 63.9 4.16 11.38 17.88 260.3 #DIV/0! 20.7 1679.5 73.9 101.4 27.5 116.1 70.4 123.3 111.9 106.0 15.63 19.32 18.61 79.6 280.1 79.9 221.5 #VALUE! 95.0 90.2 54.2 53.8 60.2 868.9 497.8 13.90 48.68 #VALUE! 125.2 229.9 729.0 100.2 145.9 166.5 1779.6 1587.8 #VALUE! 80.1 #VALUE! 113.8 35.95 #VALUE! 28.12 #VALUE! 66.8 72.3 190.0 88.1 79.7 74.9 170.4 68.8 78.6 63.3 83.1 20.52 24.74 20.67 232.9 100.4 O2 CH4 (mg/hr) (mg/hr) -0.07 -0.89 -0.12 8.34 #DIV/0! 2.83 8.53 8.29 6.56 6.88 7.60 5.50 7.72 7.92 7.54 0.62 0.60 0.57 9.05 9.45 8.94 7.35 #VALUE! 8.94 7.02 6.04 7.96 7.44 8.62 8.13 0.71 0.28 #VALUE! 7.19 6.47 7.34 6.33 6.06 5.84 6.84 7.03 #VALUE! 6.49 #VALUE! 6.08 -0.59 0.41 -0.57 1.79 6.42 5.58 6.52 7.01 6.61 6.13 6.39 6.51 10.05 7.36 7.22 -0.99 0.49 -0.59 8.45 8.28 0.00 0.00 0.00 1.84 #DIV/0! 0.98 3.54 2.57 1.42 3.11 2.40 1.48 1.86 2.54 2.06 0.00 0.00 0.00 3.12 3.43 3.14 2.67 #VALUE! 3.19 2.53 1.86 3.36 3.39 3.12 2.79 0.00 0.00 #VALUE! -0.20 2.09 2.58 2.48 1.89 1.95 3.60 -1.03 #VALUE! 2.11 #VALUE! 1.92 0.00 0.00 0.00 1.76 2.13 2.41 1.87 2.13 1.68 2.44 2.47 1.58 2.35 2.24 2.12 0.00 0.00 0.00 0.98 2.67 obs Yield #DIV/0! #DIV/0! #DIV/0! 11.44 #DIV/0! 18.03 7.03 9.52 13.52 6.45 8.53 13.54 12.80 9.75 12.15 #DIV/0! #DIV/0! #DIV/0! 6.17 6.63 7.64 7.37 #VALUE! 7.77 7.85 11.51 6.50 6.04 7.05 6.83 #DIV/0! #DIV/0! #VALUE! -103.82 10.38 7.31 9.44 12.30 12.36 5.35 -17.81 #VALUE! 9.11 #VALUE! 9.82 #DIV/0! #DIV/0! #DIV/0! 9.45 7.53 6.05 12.49 11.26 13.85 7.54 8.12 13.04 9.36 10.79 10.49 #DIV/0! #DIV/0! #DIV/0! 21.37 8.23 Cycle # Day 46 08-Dec 47 09-Dec 48 10-Dec 49 11-Dec 50 TSS OD OD (in) (out) 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1.94 2.03 2.02 2.05 2.05 2.09 2.06 1.97 1.97 1.95 1.4 1.06 1.05 2.02 2.02 1.9 1.94 1.92 1.96 1.81 1.81 1.9 2.1 2.02 1.94 1.35 0.96 0.82 2.03 2.09 2.06 2.07 2.03 2.09 1.97 1.99 2.01 1.97 1.95 1.95 1.28 0.85 0.96 2.04 2.05 2.02 1.96 1.91 1.97 2.07 1.98 1.99 2.02 1.95 2 2.9 3.18 3.11 3.16 2.77 2.68 2.89 3.07 2.97 3.19 1.48 1 1.05 2.83 2.78 2.75 3.05 3.04 3.04 2.73 2.77 2.81 3 2.79 3.04 1.42 0.99 0.92 2.97 2.97 2.81 3.19 3.25 3.18 3.06 3.02 3.05 3.06 3.06 3.11 1.45 0.97 1.09 3.03 2.96 2.96 3.18 3.2 3.18 3.03 2.87 2.91 2.96 3.08 3 457.143 547.619 519.048 528.571 342.857 280.952 395.238 523.810 476.190 590.476 38.095 -28.571 0.000 385.714 361.905 404.762 528.571 533.333 514.286 438.095 457.143 433.333 428.571 366.667 523.810 33.333 14.286 47.619 447.619 419.048 357.143 533.333 580.952 519.048 519.048 490.476 495.238 519.048 528.571 552.381 80.952 57.143 61.905 471.429 433.333 447.619 580.952 614.286 576.190 457.143 423.810 438.095 447.619 538.095 476.190 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 1.4 0.96 1.08 2.14 1.97 2.14 1.98 1.88 1.9 2.07 1.39 1.04 0.89 2.97 2.93 2.91 3.11 3.11 3.11 2.96 -4.762 38.095 -90.476 395.238 457.143 366.667 538.095 585.714 576.190 423.810 sample (mg/L) Growth Growth CO2 Gas rate rate volume Initial initial initial initial volume (day-1) (day-1) (ml) (mg/l) (mg/l) (mg/l) (mg/l) (ml) 19.25 19.5 19.5 19.5 19.75 19.75 19.75 20 20 20 23.75 23.75 23.75 23.5 23.5 23.5 23.5 23.5 23.5 23.75 23.75 23.75 23.75 23.75 23.75 23.5 23.5 23.5 23.5 23.5 23.5 23.5 23.5 23.5 23.5 23.5 23.5 23.5 23.5 23.5 18.75 18.75 18.75 19 19 19 19.25 19.25 19.25 19.5 19.5 19.5 20 20 20 9.89 10.87 10.46 10.49 7.26 6.01 8.15 10.48 9.72 11.58 1.12 -1.18 0.00 6.82 6.47 7.47 9.09 9.22 8.82 8.19 8.47 7.81 7.13 6.47 8.93 1.03 0.63 2.35 7.68 7.10 6.29 8.70 9.44 8.45 8.85 8.40 8.40 8.85 9.05 9.37 3.19 3.38 3.25 9.87 9.18 9.54 11.84 12.59 11.72 9.27 9.03 9.24 9.06 10.78 9.60 0.49 0.54 0.52 0.52 0.36 0.30 0.41 0.52 0.49 0.58 0.06 -0.06 0.00 0.34 0.32 0.37 0.45 0.46 0.44 0.41 0.42 0.39 0.36 0.32 0.45 0.05 0.03 0.12 0.38 0.36 0.31 0.43 0.47 0.42 0.44 0.42 0.42 0.44 0.45 0.47 0.16 0.17 0.16 0.49 0.46 0.48 0.59 0.63 0.59 0.46 0.45 0.46 0.45 0.54 0.48 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 432.9 436.3 443.4 429.8 431.5 437.7 #VALUE! 429.6 428.3 #VALUE! 481.1 471.7 486.2 391.9 394.9 409.4 406.4 405.5 396.1 394.5 399.8 382.9 375.6 390.3 378.4 525.0 522.2 #VALUE! 516.3 499.0 #VALUE! 502.1 503.6 502.0 506.6 500.0 #VALUE! 501.3 508.0 488.0 529.6 516.5 #VALUE! 495.2 484.3 490.6 475.2 460.9 #VALUE! 466.9 454.7 #VALUE! 464.8 459.8 #VALUE! 436.2 424.2 409.8 452.3 450.1 407.8 #VALUE! 429.2 419.9 #VALUE! 733.3 752.6 742.1 485.5 501.2 474.7 476.7 464.1 509.0 528.8 484.0 509.7 527.4 500.6 530.6 347.8 401.8 #VALUE! 239.0 299.3 #VALUE! 274.8 250.5 275.1 253.7 302.0 #VALUE! 273.6 255.4 294.6 405.6 397.7 #VALUE! 383.0 295.8 286.4 306.9 351.0 #VALUE! 337.1 373.4 #VALUE! 323.5 341.2 #VALUE! 200.0 202.8 213.8 183.7 192.1 205.2 #VALUE! 195.8 199.8 #VALUE! 0.0 0.0 0.0 197.3 184.0 178.4 186.9 198.1 174.0 165.9 184.0 175.8 166.6 177.1 163.7 0.0 0.0 0.0 278.7 259.2 #VALUE! 267.9 276.1 268.8 279.1 254.5 #VALUE! 268.9 278.7 259.0 0.0 0.0 0.0 126.5 264.2 268.1 261.8 240.4 #VALUE! 248.2 231.1 #VALUE! 256.1 248.7 #VALUE! 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 46.3 44.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 50.7 46.3 #VALUE! 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 53.4 73.4 #VALUE! 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 73.4 81.9 20.67 20.67 20.67 20.92 20.92 20.92 21.2 21.2 21.2 21.42 -0.17 1.86 -4.48 7.45 8.99 7.00 10.05 11.16 10.94 7.93 -0.01 0.09 -0.22 0.37 0.45 0.35 0.50 0.56 0.55 0.40 400 400 400 400 400 400 400 400 400 400 558.4 558.3 546.0 502.9 504.6 504.3 503.7 493.8 489.4 487.1 429.9 417.5 433.6 257.3 268.2 256.2 271.6 261.1 288.7 285.7 0.0 0.0 0.0 273.4 269.5 276.5 271.3 276.3 255.0 271.4 0.0 0.0 #VALUE! 0.0 0.0 0.0 0.0 0.0 0.0 0.0 time(hr) inc Gas O2 N2 CH4 O2 final N2 final CO2 O2 % O2 CH4 % CH4 CO2 CH4 obs ed (mg) ed ed (mg) ed d (mg) 755.6 471.0 406.8 461.9 283.1 494.9 276.4 200.6 184.0 #VALUE! 690.3 685.0 #VALUE! 714.3 747.0 #VALUE! 742.6 316.5 #VALUE! 133.6 440.9 #VALUE! 796.6 #VALUE! 653.7 319.6 311.5 #VALUE! 407.5 116.3 #VALUE! 474.3 413.1 #VALUE! 455.1 390.2 #VALUE! 497.1 529.6 484.3 293.3 246.5 #VALUE! 234.9 108.2 542.9 536.1 135.4 317.5 490.7 452.9 #VALUE! 133.4 596.3 #VALUE! 13.1 86.5 73.6 82.9 54.2 196.6 49.5 33.7 42.0 #VALUE! 0.0 0.0 0.0 107.2 108.1 #VALUE! 117.2 46.6 #VALUE! 12.8 79.1 #VALUE! 106.6 #VALUE! 84.6 0.0 0.0 0.0 156.9 50.6 #VALUE! 205.9 218.3 #VALUE! 209.7 239.0 #VALUE! 200.6 181.3 177.9 0.0 0.0 #VALUE! 89.6 33.3 183.6 195.0 43.9 98.5 172.5 225.2 #VALUE! 25.5 184.7 #VALUE! 277.2 289.3 291.0 288.4 227.0 152.2 245.2 313.3 212.7 #VALUE! 72.54 62.30 #VALUE! 293.3 258.5 #VALUE! 220.3 217.2 #VALUE! 204.7 162.4 #VALUE! 240.7 #VALUE! 302.6 101.02 89.45 #VALUE! 342.7 320.4 #VALUE! 290.1 276.3 #VALUE! 265.7 282.6 #VALUE! 397.4 353.8 316.0 103.24 141.51 #VALUE! 392.0 384.5 353.8 354.2 323.5 326.6 295.9 239.4 #VALUE! 380.9 369.8 #VALUE! 162.02 148.12 150.97 142.36 120.49 75.26 #VALUE! 152.74 150.17 #VALUE! 1.56 -11.08 #VALUE! 139.86 157.95 #VALUE! 150.06 145.18 #VALUE! 157.81 121.46 #VALUE! 150.24 #VALUE! 144.36 -0.23 -38.54 #VALUE! 167.50 199.59 #VALUE! 184.25 188.46 #VALUE! 179.58 118.36 #VALUE! 191.08 197.05 187.92 26.50 35.38 #VALUE! 170.39 173.19 178.55 164.48 152.61 #VALUE! 149.37 102.27 #VALUE! 162.40 173.12 #VALUE! 93.6 84.9 85.1 82.8 69.8 43.0 #VALUE! 88.9 87.6 #VALUE! 0.8 -5.9 #VALUE! 89.2 100.0 #VALUE! 92.3 89.5 #VALUE! 100.0 75.9 #VALUE! 100.0 #VALUE! 95.4 -0.1 -18.5 #VALUE! 81.1 100.0 #VALUE! 91.7 93.6 #VALUE! 88.6 59.2 #VALUE! 95.3 97.0 96.3 12.5 17.1 #VALUE! 86.0 89.4 91.0 86.5 82.8 #VALUE! 80.0 56.2 #VALUE! 87.3 94.1 #VALUE! 76.96 49.98 55.88 41.00 42.34 17.29 #VALUE! 49.46 41.58 #VALUE! 0.00 0.00 #VALUE! 49.79 44.59 #VALUE! 44.64 51.93 #VALUE! 46.04 38.86 #VALUE! 38.43 #VALUE! 37.99 0.00 0.00 #VALUE! 74.68 51.55 #VALUE! 59.45 57.49 #VALUE! 64.90 27.81 #VALUE! 63.38 76.53 60.30 0.00 0.00 #VALUE! -7.81 69.32 68.48 60.07 50.63 #VALUE! 51.88 18.21 #VALUE! 77.75 57.21 #VALUE! 96.2 61.6 65.3 55.8 55.1 21.1 #VALUE! 63.1 52.0 #VALUE! #DIV/0! #DIV/0! #VALUE! 63.1 60.6 #VALUE! 59.7 65.5 #VALUE! 69.4 52.8 #VALUE! 57.7 #VALUE! 58.0 #DIV/0! #DIV/0! #VALUE! 67.0 49.7 #VALUE! 55.5 52.1 #VALUE! 58.1 27.3 #VALUE! 58.9 68.6 58.2 #DIV/0! #DIV/0! #VALUE! -15.4 65.6 63.9 57.4 52.7 #VALUE! 52.3 19.7 #VALUE! 75.9 57.5 #VALUE! 64.0 104.2 117.3 113.0 144.3 50.2 #VALUE! 268.1 194.2 #VALUE! 12.31 9.58 #VALUE! 79.7 69.4 #VALUE! 56.6 127.4 #VALUE! 324.2 71.3 #VALUE! 63.8 #VALUE! 98.3 23.67 27.64 #VALUE! 80.4 329.7 #VALUE! 67.2 67.0 #VALUE! 59.2 87.5 #VALUE! 87.5 68.2 76.9 35.74 61.95 #VALUE! 255.7 420.4 74.7 81.1 335.5 #VALUE! 81.3 78.9 #VALUE! 369.5 55.3 #VALUE! 8.42 7.60 7.74 7.30 6.10 3.81 #VALUE! 7.64 7.51 #VALUE! 0.07 -0.47 #VALUE! 5.95 6.72 #VALUE! 6.39 6.18 #VALUE! 6.64 5.11 #VALUE! 6.33 #VALUE! 6.08 -0.01 -1.64 #VALUE! 7.13 8.49 #VALUE! 7.84 8.02 #VALUE! 7.64 5.04 #VALUE! 8.13 8.39 8.00 1.41 1.89 #VALUE! 8.97 9.12 9.40 8.54 7.93 #VALUE! 7.66 5.24 #VALUE! 8.12 8.66 #VALUE! 4.00 2.56 2.87 2.10 2.14 0.88 #VALUE! 2.47 2.08 #VALUE! 0.00 0.00 #VALUE! 2.12 1.90 #VALUE! 1.90 2.21 #VALUE! 1.94 1.64 #VALUE! 1.62 #VALUE! 1.60 0.00 0.00 #VALUE! 3.18 2.19 #VALUE! 2.53 2.45 #VALUE! 2.76 1.18 #VALUE! 2.70 3.26 2.57 0.00 0.00 #VALUE! -0.41 3.65 3.60 3.12 2.63 #VALUE! 2.66 0.93 #VALUE! 3.89 2.86 #VALUE! 5.94 10.96 9.29 12.89 8.10 16.25 #VALUE! 10.59 11.45 #VALUE! #DIV/0! #DIV/0! #VALUE! 7.75 8.12 #VALUE! 11.84 10.27 #VALUE! 9.52 11.77 #VALUE! 11.15 #VALUE! 13.79 #DIV/0! #DIV/0! #VALUE! 5.99 8.13 #VALUE! 8.97 10.11 #VALUE! 8.00 17.64 #VALUE! 8.19 6.91 9.16 #DIV/0! #DIV/0! #VALUE! -60.35 6.25 6.54 9.67 12.13 #VALUE! 8.81 23.28 #VALUE! 5.76 9.40 #VALUE! 414.4 400.4 426.9 463.4 485.1 433.6 526.0 474.5 516.3 452.6 0.0 0.0 #VALUE! 201.8 195.3 196.4 200.7 208.4 200.3 236.3 88.11 97.01 98.79 400.5 401.8 379.6 325.3 291.5 314.6 293.3 10.35 8.78 9.58 184.87 185.76 190.54 181.13 181.05 175.26 152.65 4.6 3.9 4.4 91.9 92.0 94.5 89.9 91.7 89.5 78.3 0.00 0.00 #VALUE! 64.55 64.60 64.21 67.07 64.62 57.21 48.90 #DIV/0! #DIV/0! #VALUE! 59.0 59.9 58.0 61.8 58.5 56.1 45.0 36.57 40.46 #VALUE! 89.0 88.9 89.7 67.2 64.2 70.4 74.1 0.50 0.42 0.46 8.84 8.88 9.11 8.54 8.54 8.27 7.13 0.00 0.00 #VALUE! 3.09 3.09 3.07 3.16 3.05 2.70 2.28 #DIV/0! #DIV/0! #VALUE! 6.12 7.08 5.71 8.02 9.06 10.07 8.67 231 360 403 392 636 330 #VALUE! 856 913 #VALUE! 424.9 439.5 #VALUE! 272 268 #VALUE! 257 586 #VALUE! 1584 439 #VALUE! 265 #VALUE! 325 435.2 516.0 #VALUE! 235 1029 #VALUE! 232 242 #VALUE! 223 310 #VALUE! 220 193 243 553.1 645.3 #VALUE! 652 1093 211 229 1037 #VALUE! 275 330 #VALUE! 970 229 #VALUE! 48.3 73.3 65.5 75.4 82.0 302.8 67.1 22.3 23.2 #VALUE! 449.2 454.5 #VALUE! 62.1 0.0 #VALUE! 48.7 29.0 #VALUE! 0.0 87.6 #VALUE! 0.0 #VALUE! 21.6 483.0 479.5 #VALUE! 166.4 0.0 #VALUE! 71.7 53.5 #VALUE! 103.4 263.7 #VALUE! 42.8 31.9 29.9 335.1 265.3 #VALUE! 42.5 18.8 83.9 111.9 30.6 73.3 136.1 241.4 #VALUE! 24.2 47.3 #VALUE! 415.0 417.1 406.3 222 221 236 207 220 224 253 513.3 514.4 514.0 73.3 72.7 47.3 98.6 74.9 91.7 167.1 consum Consum consum Consum Produce O2 final (mg/l) (mg/l) 78 CH4 final (mg/l) (mg/l) (mg/hr) (mg/hr) Yield Cycle # Day sample 4-2 4-3 5-1 5-2 5-3 OD OD (in) (out) 1.62 1.86 1.89 2.04 1.97 2.48 2.87 2.97 3.1 3.04 TSS Growth Growth Gas (mg/L) time(hr) rate rate volume inc 409.524 480.952 514.286 504.762 509.524 21.42 21.42 21.42 21.42 21.42 O2 N2 CH4 CO2 Gas Initial initial initial initial volume (day-1) (day-1) (ml) (mg/l) (mg/l) (mg/l) (mg/l) (ml) 9.40 9.57 9.96 9.24 9.57 0.47 0.48 0.50 0.46 0.48 400 400 400 400 400 487.5 482.0 467.3 475.4 477.5 301.3 308.1 350.8 304.6 298.9 261.5 255.6 237.1 258.9 263.9 0.0 0.0 0.0 0.0 0.0 314 262 263 276 208 O2 final N2 final (mg/l) (mg/l) 331.3 115.6 37.3 31.5 57.9 384.0 470.0 533.7 441.1 574.8 79 CH4 CO2 final final O2 % O2 CH4 % CH4 CO2 (mg/l) (mg/l) ed (mg) ed ed (mg) ed d (mg) 242.2 175.6 197.0 152.3 190.9 111.7 227.8 319.1 372.9 373.8 91.00 162.48 177.09 181.47 178.98 46.7 84.3 94.7 95.4 93.7 28.62 56.18 43.04 61.48 65.87 27.4 55.0 45.4 59.4 62.4 35.1 59.8 83.9 103.0 77.7 consum Consum consum Consum Produce O2 CH4 (mg/hr) (mg/hr) 4.25 7.59 8.27 8.47 8.36 1.34 2.62 2.01 2.87 3.08 obs Yield 14.31 8.56 11.95 8.21 7.74 APPENDIX B Data Analysis for COD Consumption and Nitrogen Balance: Cycle # Day sample CO2 Produce d (mg) O2 (mg/hr) 23-Oct 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 -5.03 4.13 64.53 49.4 25.0 20.1 81.3 735.7 52.9 58.0 54.8 63.9 86.1 70.9 101.1 10.55 69.70 53.90 64.7 161.5 81.0 62.8 71.6 54.4 81.8 35.9 76.5 116.1 89.4 106.1 12.99 15.08 30.80 48.6 60.5 32.8 82.6 88.3 87.0 62.3 79.4 63.8 73.1 73.0 43.3 15.21 47.13 45.88 177.6 116.2 83.5 83.6 62.1 84.7 119.0 98.8 60.9 80.6 52.7 0.66 0.52 2.64 5.97 7.68 4.74 8.36 9.27 8.35 8.04 7.96 8.16 8.31 8.61 7.17 1.24 0.77 0.90 7.24 8.92 7.94 10.21 10.69 10.48 9.49 10.63 9.42 11.17 10.55 9.93 0.28 0.54 0.53 8.12 7.95 7.33 7.39 7.30 7.51 7.13 7.16 7.67 8.37 8.11 9.43 0.89 0.87 1.21 8.56 9.18 9.03 13.60 5.08 8.80 8.24 8.79 8.15 9.36 9.05 1 24-Oct 2 25-Oct 3 26-Oct 4 CH4 (mg/hr) 0.00 0.00 0.00 2.01 3.35 1.58 2.70 3.20 3.11 2.94 2.82 3.04 2.90 2.69 2.09 0.00 0.00 0.00 2.10 3.49 2.49 3.44 3.98 4.02 3.28 4.44 3.40 4.09 3.57 3.00 0.00 0.00 0.00 3.85 2.78 3.35 2.37 2.28 2.41 2.19 2.21 2.35 3.10 2.78 3.74 0.00 0.00 0.00 3.24 1.56 3.38 2.23 1.54 3.70 3.11 2.71 4.03 3.29 4.20 obs Yield #DIV/0! #DIV/0! #DIV/0! 7.69 3.14 7.39 6.61 5.88 5.30 6.88 7.33 6.66 6.36 7.75 8.82 #DIV/0! #DIV/0! #DIV/0! 8.00 6.47 8.45 7.22 6.30 6.86 5.88 4.86 5.53 6.37 9.12 11.71 #DIV/0! #DIV/0! #DIV/0! 4.15 6.26 3.77 8.31 8.26 7.56 7.62 7.75 8.00 6.15 6.09 4.64 #DIV/0! #DIV/0! #DIV/0! 5.29 10.53 4.37 9.40 13.91 5.41 6.06 6.42 4.67 6.52 4.03 COD (mg/L) IN 411 411 411 0 0 0 404 404 404 225 225 225 626 626 626 411 411 411 0 0 0 404 404 404 225 225 225 626 626 626 411 411 411 0 0 0 404 404 404 225 225 225 626 626 626 411 411 411 0 0 0 404 404 404 225 225 225 626 626 NH4- N (mg/L ) IN (Cal) 53 53 53 56 56 56 53 53 53 30 30 30 76 76 76 53 53 53 56 56 56 53 53 53 30 30 30 76 76 76 53 53 53 56 56 56 53 53 53 30 30 30 76 76 76 53 53 53 56 56 56 53 53 53 30 30 30 76 76 COD COD: (mg/L) N OUT 7.7 7.7 7.7 0.0 0.0 0.0 7.6 7.6 7.6 7.5 7.5 7.5 8.2 8.2 8.2 7.7 7.7 7.7 0.0 0.0 0.0 7.6 7.6 7.6 7.5 7.5 7.5 8.2 8.2 8.2 7.7 7.7 7.7 0.0 0.0 0.0 7.6 7.6 7.6 7.5 7.5 7.5 8.2 8.2 8.2 7.7 7.7 7.7 0.0 0.0 0.0 7.6 7.6 7.6 7.5 7.5 7.5 8.2 8.2 75 65 58 168 168 180 193 115 149 141 154 159 92 90 158 56 59 123 111 127 127 80 87 92 106 100 117 90 101 126 0 0 0 49 39 42 76 67 44 45 57 30 15 96 55 22 52 30 69 45 50 63 60 62 65 60 83 76 40 NH4(mg/L) OUT (Cal) 14 39 53 0 0 0 0 0 0 0 0 0 0 0 0 62 49 52 52 16 21 0 0 0 0 0 0 0 0 0 62 46 4 62 6 10 13 0 0 0 0 0 0 0 0 63 67 65 16 11 13 0 0 0 0 0 0 0 0 NH4-N (mg/L) OUT COD (mg/L) Consume d % COD Consume d NH4-N (mg/L) Consum ed 11.14 30.25 41.71 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 48.05 38.22 40.73 40.84 12.34 16.38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 48.38 36.04 3.28 48.59 4.94 8.07 10.12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 49.47 52.20 50.34 12.56 8.74 10.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 336 346 353 -168 -168 -180 211 289 255 84 71 66 534 536 468 355 352 288 -111 -127 -127 324 317 312 119 125 108 536 525 500 411 411 411 -49 -39 -42 328 337 360 180 168 195 611 530 571 389 359 381 -69 -45 -50 341 344 342 160 165 142 550 586 82% 84% 86% #DIV/0! #DIV/0! #DIV/0! 52% 72% 63% 37% 32% 29% 85% 86% 75% 86% 86% 70% #DIV/0! #DIV/0! #DIV/0! 80% 78% 77% 53% 56% 48% 86% 84% 80% 100% 100% 100% #DIV/0! #DIV/0! #DIV/0! 81% 83% 89% 80% 75% 87% 98% 85% 91% 95% 87% 93% #DIV/0! #DIV/0! #DIV/0! 84% 85% 85% 71% 73% 63% 88% 94% 42.34 23.23 11.77 55.72 55.72 55.72 53.40 53.40 53.40 30.10 30.10 30.10 76.44 76.44 76.44 5.43 15.26 12.75 14.88 43.38 39.34 53.40 53.40 53.40 30.10 30.10 30.10 76.44 76.44 76.44 5.10 17.44 50.20 7.13 50.78 47.65 43.27 53.40 53.40 30.10 30.10 30.10 76.44 76.44 76.44 4.01 1.28 3.14 43.16 46.98 45.67 53.40 53.40 53.40 30.10 30.10 30.10 76.44 76.44 80 NO3(mg/L) NO2(mg/L) 1.7 1.97 1.43 0.18 0 0 0 0 0 0 0 0.34 0.2 0 0 1.9 1.6 1.58 0.2 0 0 0 0 0.26 0 0 0 0.1 0 0 1.2 1.15 1.29 0 0 0.17 0.09 0.11 0.11 0.11 0 0 0.11 0.19 0.12 1.68 1.44 1.12 0 0 0 0.14 0.26 0 0.13 0.22 0.14 0.11 0.11 1.8 1.3 0.78 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.12 0 0 0 0 0 0 0 0 0 NH4-N NH4-N Total Organic for Cell for Nitrogen Nitrogen Synthesis nitrificat n OUT (mg/l) 0.12 TSS ion (mg/l) -4.3 46.6 -1.7 24.9 0.0 11.8 44.6 11.1 30.3 25.4 33.7 22.0 51.4 2.0 54.3 -0.9 47.4 6.0 58.3 -28.2 59.4 -29.3 58.3 -28.2 53.1 23.3 60.0 16.4 53.1 23.3 5.1 0.3 4.6 10.7 12.0 0.7 38.3 -23.4 51.4 -8.0 48.0 -8.7 56.6 -3.2 57.1 -3.7 62.9 -9.5 44.0 -13.9 49.1 -19.0 42.9 -12.8 59.4 17.0 74.3 2.2 80.0 -3.6 14.3 -9.2 13.7 3.7 17.1 33.1 44.0 -36.9 48.0 2.8 34.9 12.8 54.3 -11.0 52.0 1.4 50.3 3.1 45.1 -15.0 46.3 -16.2 50.9 -20.8 51.4 25.0 45.7 30.7 46.9 29.6 12.0 -8.0 -36.6 37.9 4.6 -1.4 41.1 2.0 39.4 7.6 35.4 10.2 50.3 3.1 51.4 2.0 48.0 5.4 45.1 -15.0 41.7 -11.6 45.1 -15.0 51.4 25.0 40.6 35.9 Nitrogen NH4-N% N% for Total Convert converted Cell Nitrogen ed to gas to gas Synthes n IN (mg/l) is (mg/l) NH4-N% converted to biomass Cycle # Day 27-Oct 5 28-Oct 6 29-Oct 7 30-Oct 8 NH4-N (mg/L) OUT COD (mg/L) Consume d % COD Consume d NH4-N (mg/L) Consum ed 0 77 53 49 19 13 14 0 0 0 0 0 0 0 0 0 98 69 87 77 61 68 0 0 0 0 0 0 24 0 0 93 91 78 74 84 86 73 0 0 0 0 0 42 14 18 95 88 97 0.00 60.17 41.28 38.00 14.85 9.94 10.81 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 76.55 54.16 67.49 10.00 5.00 15.00 0.00 0.00 0.00 0.00 0.00 0.00 18.78 0.00 0.00 72.19 70.65 60.82 12.00 5.00 3.00 57.00 0.00 0.00 0.00 0.00 0.00 32.98 10.75 14.31 74.27 68.25 75.39 571 364 386 371 -233 -38 -201 304 349 374 131 154 149 567 586 571 337 344 339 -158 -158 -155 278 296 288 50 -14 99 515 487 493 356 356 353 -405 -458 -522 274 292 249 -195 -353 -347 528 516 506 358 361 359 91% 89% 94% 90% #DIV/0! #DIV/0! #DIV/0! 75% 86% 93% 58% 68% 66% 91% 94% 91% 91% 92% 91% -263% -263% -258% 75% 80% 77% 28% -8% 56% 92% 87% 88% 96% 96% 95% -675% -763% -870% 74% 78% 67% -111% -201% -197% 94% 92% 90% 96% 97% 97% 76.44 -6.69 12.20 15.48 40.87 45.78 44.91 53.40 53.40 53.40 30.10 30.10 30.10 76.44 76.44 76.44 -19.95 2.44 -10.89 44.00 49.00 39.00 62.00 62.00 62.00 33.00 33.00 33.00 66.22 85.00 85.00 -15.59 -14.05 -4.22 44.60 51.60 53.60 -0.40 56.60 56.60 33.00 33.00 33.00 52.02 74.25 70.69 -17.67 -11.65 -18.79 0.11 1.47 1.41 1.17 0.18 0.08 0 0.11 0.08 0.09 0 0 0.1 0.12 0.11 0.16 1.2 1.14 1.17 0.73 0.7 0.17 0.17 0.155 0.09 0.18 0 0.22 0.142 0.39 0.15 0.932 0.932 0.934 0.792 0.904 0.72 0.48 0.1 0.1 0 0.12 0.11 0.105 0.094 0.113 0.68 0.64 0.72 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 51.4 -34.9 -12.0 -34.9 13.1 20.6 22.3 52.0 57.7 78.3 52.0 54.3 49.7 72.0 65.7 74.3 -13.7 -2.9 -5.7 20.0 29.1 22.3 57.7 59.4 56.0 47.4 44.6 45.7 44.0 54.9 57.7 -12.0 -13.1 -14.9 33.1 22.9 15.4 24.6 60.0 47.4 39.4 32.0 39.4 48.6 57.7 58.3 -3.4 -1.1 4.0 25.0 28.2 24.2 50.3 27.7 25.2 22.6 1.4 -4.3 -24.9 -21.9 -24.2 -19.6 4.4 10.7 2.2 -6.2 5.3 -5.2 24.0 19.9 16.7 4.3 2.6 6.0 -14.4 -11.6 -12.7 22.2 30.1 27.3 -3.6 -0.9 10.6 11.5 28.7 38.2 -25.0 -3.4 9.2 -6.4 1.0 -6.4 3.5 16.5 12.4 -14.2 -10.5 -22.8 84 342 544 35 197 77 172 76 48 55 82 0 0 0 0 0 10.00 5.00 3.00 0.00 0.00 0.00 0.00 0.00 -24 -282 -484 337 175 295 4 100 -40% -470% -807% 91% 47% 79% 2% 57% 46.60 51.60 53.60 56.60 56.60 56.60 33.00 33.00 0.405 0.71 0.893 0.152 0 0 0.092 0.083 0 0 0 0 0 0 0 0 42.3 40.6 29.1 71.4 42.9 42.9 32.6 44.6 4.3 11.0 24.5 -14.8 13.7 13.7 0.4 -11.6 488 27 0 20 0.00 15.46 -312 535 -177% 95% 33.00 69.54 2.5 0.115 0 0 20.0 55.4 13.0 14.1 CO2 Produce d (mg) O2 (mg/hr) 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 1-1 1-2 1-3 95.7 17.14 68.56 48.10 79.6 47.0 69.2 119.7 205.7 81.2 79.1 63.9 57.5 95.7 96.5 78.7 5.95 7.35 33.72 81.7 78.4 75.9 77.8 81.8 91.7 106.0 84.8 60.3 82.0 84.1 117.0 27.82 38.56 68.64 204.9 73.4 72.2 62.1 79.8 153.6 88.7 77.5 86.9 71.4 113.5 130.0 #VALUE! #VALUE! #VALUE! 8.58 0.90 -1.18 1.64 8.36 5.70 8.20 9.05 8.82 8.48 6.64 5.88 6.61 8.78 9.43 5.76 0.68 0.74 0.71 7.21 7.73 8.44 10.06 9.94 9.25 9.12 9.18 8.88 9.42 10.11 9.26 0.57 0.66 0.54 8.53 5.40 5.30 2.99 9.91 9.22 9.48 6.84 8.32 9.88 10.24 10.86 0.74 7.78 0.11 7.23 #DIV/0! #DIV/0! #DIV/0! 1.68 2.37 2.96 5.46 6.16 9.65 9.24 11.07 8.88 7.83 6.45 14.40 #DIV/0! #DIV/0! #DIV/0! 3.59 4.74 3.44 6.55 6.90 7.79 5.98 5.87 4.91 5.85 6.53 7.81 #DIV/0! #DIV/0! #DIV/0! 4.12 5.22 3.51 14.72 6.78 6.06 4.20 5.53 6.41 5.11 5.55 6.22 #DIV/0! #DIV/0! #DIV/0! 626 411 411 411 0 0 0 404 404 404 225 225 225 626 626 626 372 372 372 60 60 60 372 372 372 176 176 176 562 562 562 372 372 372 60 60 60 372 372 372 176 176 176 562 562 562 372 372 372 76 53 53 53 56 56 56 53 53 53 30 30 30 76 76 76 57 57 57 54 54 54 62 62 62 33 33 33 85 85 85 57 57 57 57 57 57 57 57 57 33 33 33 85 85 85 57 57 57 8.2 7.7 7.7 7.7 0.0 0.0 0.0 7.6 7.6 7.6 7.5 7.5 7.5 8.2 8.2 8.2 6.6 6.6 6.6 1.1 1.1 1.1 6.0 6.0 6.0 5.3 5.3 5.3 6.6 6.6 6.6 6.6 6.6 6.6 1.1 1.1 1.1 6.6 6.6 6.6 5.3 5.3 5.3 6.6 6.6 6.6 6.6 6.6 6.6 55 47 25 40 233 38 201 100 55 30 94 71 76 59 40 55 35 28 33 218 218 215 94 76 84 126 190 77 47 75 69 16 16 19 465 518 582 98 80 123 371 529 523 34 46 56 14 11 13 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 #VALUE! 9.08 2.85 6.87 #VALUE! 10.23 3.47 5.41 #VALUE! 6.76 2.09 6.47 #VALUE! 9.26 3.55 9.31 #VALUE! 8.53 2.49 7.98 #VALUE! 9.38 3.02 6.56 #VALUE! 9.09 3.95 3.82 #VALUE! #VALUE! #VALUE #VALUE ! ! #VALUE! 5.75 1.95 4.76 #VALUE! 9.94 3.69 6.96 60 60 60 372 372 372 176 176 57 57 57 57 57 57 33 33 1.1 1.1 1.1 6.6 6.6 6.6 5.3 5.3 176 562 33 85 5.3 6.6 4-3 5-1 CH4 (mg/hr) 2.96 0.00 0.00 0.00 2.84 3.15 2.72 3.45 3.40 2.94 2.04 1.78 2.03 3.33 3.69 1.87 0.00 0.00 0.00 2.32 2.56 2.70 3.67 3.59 2.99 3.31 3.17 3.88 3.14 3.50 3.08 0.00 0.00 0.00 3.27 1.78 1.79 0.68 3.60 3.18 3.81 2.35 2.50 3.87 4.23 3.81 0.00 0.00 0.00 obs Yield COD (mg/L) IN NH4N (mg/L ) IN (Cal) sample COD COD:N (mg/L) OUT NH4(mg/L) OUT (Cal) 81 NO3(mg/L) NO2(mg/L) NH4-N for Cell Synthesi s 0.12 TSS NH4-N Total Organic for Nitroge n Nitroge nitrifica OUT n(mg/l) tion (mg/l) Nitroge NH4-N% NH4-N% n converte d converte Conver d to to gas ted to biomass gas (mg/l) N% for Cell Synthes is Total Nitroge n IN (mg/l) 144.3 144.3 140.8 12% 11% 11% 178.9 171.1 174.8 24 21 19 129.8 129.9 89.8 85.0 87.8 145.1 183.6 175.9 8% 8% 6% 6% 6% 10% 11% 11% 151.5 153.9 99.2 96.8 97.1 194.7 216.3 206.5 139 140 144 132 144 95 76 128.6 134.3 140.1 10% 10% 12% 132.0 144.0 94.9 75.9 95 177 92.5 161.4 155 150 156 130 130 90 85 88 164 184 176 35.8 50.3 37.1 22 24 9 12 9 31 32 31 44.3 39.2 34.8 0.0 34.7 38.6 27.7 35.8 27.4 36.1 38.0 35.9 151.2 155.6 167.5 12 16 24 21.6 27.5 41.6 60.0 62.4 51.5 9% 10% 7% 6% 156.1 166.5 109.4 87.5 24 23 14 11 42.7 39.8 43.6 34.7 57.3 60.2 56.1 65.0 8% 11% 112.7 197.5 18 20 53.5 24.1 38.9 57.6 65.0 61.3 71.7 64.2 71.9 41.6 61.5 63.9 - Cycle # sample CO2 Produce d (mg) O2 (mg/hr) 01-Nov 5-2 5-3 1-1 86.8 96.0 41.55 9.79 9.24 0.54 3.59 3.13 0.00 6.71 7.52 #DIV/0! 562 562 372 85 85 57 6.6 6.6 6.6 28 35 22 1-2 25.80 1.85 0.00 #DIV/0! 372 57 6.6 16 1-3 18.62 1.80 0.00 #DIV/0! 372 57 6.6 18 2-1 303.0 7.39 2.65 5.93 60 57 1.1 81 2-2 180.9 6.95 2.30 5.31 60 57 1.1 103 2-3 257.7 6.88 2.22 6.42 60 57 1.1 66 3-1 190.0 7.86 2.66 8.09 372 57 6.6 70 3-2 62.0 4.69 2.09 8.21 372 57 6.6 58 3-3 85.3 6.02 1.85 5.37 372 57 6.6 428 4-1 318.7 5.50 1.40 5.02 176 33 5.3 564 4-2 103.3 6.19 2.23 2.04 176 33 5.3 517 4-3 222.1 8.02 2.82 6.31 176 33 5.3 103 5-1 214.2 8.41 3.55 5.66 562 85 6.6 39 5-2 214.8 8.37 3.07 6.27 562 85 6.6 42 5-3 92.6 7.95 2.46 7.49 562 85 6.6 38 1-1 21.74 3.00 0.00 #DIV/0! 372 57 6.6 21 1-2 40.65 0.74 0.00 #DIV/0! 372 57 6.6 20 1-3 0.00 0.79 0.00 #DIV/0! 372 57 6.6 20 2-1 92.8 8.63 3.54 6.38 60 57 1.1 62 2-2 76.6 8.26 1.88 9.06 60 57 1.1 102 2-3 262.4 7.21 3.91 3.34 60 57 1.1 50 3-1 63.6 8.37 2.71 8.04 372 57 6.6 188 3-2 79.6 8.34 2.70 9.18 372 57 6.6 76 3-3 67.3 8.62 3.09 8.27 372 57 6.6 74 4-1 100.2 8.49 3.20 6.67 176 33 5.3 102 4-2 59.3 7.65 2.61 8.07 176 33 5.3 118 4-3 57.2 7.64 2.53 5.46 176 33 5.3 503 5-1 81.6 8.23 1.78 13.41 562 85 6.6 29 5-2 65.0 8.73 2.66 9.79 562 85 6.6 31 5-3 65.2 8.48 2.67 9.76 562 85 6.6 43 10 02-Nov 11 CH4 (mg/hr) obs Yield COD (mg/L) IN NH4N (mg/L ) IN (Cal) Day COD:N COD (mg/L) OUT NH4(mg/L) OUT (Cal) NH4-N (mg/L) OUT 12 9.31 22 17.17 #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! COD (mg/L) Consume d % COD Consume d NH4-N (mg/L) Consum ed 534 527 350 95% 94% 94% 356 96% 354 95% -21 -35% -43 -72% -6 -10% 302 81% 314 84% -56 -15% -388 -220% -341 -194% 73 41% 523 93% 520 93% 524 93% 351 94% 352 95% 352 95% -2 -3% -42 -70% 10 17% 184 49% 296 80% 298 80% 74 42% 58 33% -327 -186% 533 95% 531 94% 519 92% 75.69 67.83 #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 82 NO3(mg/L) NO2(mg/L) NH4-N for Cell Synthesi s 0.12 TSS 0 0.103 - 0 0 0 52.0 50.9 2.9 - 0 -1.1 - 0 -1.1 - 0 43.4 - 0 33.7 - 0 39.4 - 0 59.4 - 0 47.4 - 0 27.4 - 0 19.4 - 0 12.6 - 0 49.1 - 0 55.4 - 0 53.1 - 0 50.9 - 0 -1.7 - 0 0.6 - 0 4.0 - 0 51.4 - 0 38.9 - 0 29.7 - 0 49.7 - 0 56.6 - 0 58.3 - 0 48.6 - 0 48.0 - 0 31.4 - 0 54.3 - 0 59.4 - 0 59.4 NH4-N Total Organic for Nitroge n Nitroge nitrifica OUT n(mg/l) tion (mg/l) 23.7 17.0 #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 170 175 160.7 157.7 N% for Cell Synthes is Total Nitroge n IN (mg/l) 10% 10% 201.2 200.1 Nitroge NH4-N% NH4-N% n converte d converte Conver d to to gas ted to biomass gas (mg/l) 31 25 36.8 29.5 52.3 50.2 Cycle # CO2 Produce d (mg) O2 (mg/hr) 28-Nov 1-1 30.05 1.75 0.00 #DIV/0! 354 65 5.4 13 1-2 83.62 1.22 0.00 #DIV/0! 354 65 5.4 - 1-3 #DIV/0! #DIV/0! #DIV/0! #DIV/0! 354 65 5.4 - 2-1 214.5 6.96 2.87 7.52 10 56 0.2 25 2-2 #DIV/0! #DIV/0! #DIV/0! #DIV/0! 10 56 0.2 - 2-3 112.0 7.21 2.08 10.46 10 56 0.2 - 3-1 219.7 7.74 3.32 8.03 341 55 6.2 - 3-2 71.4 7.48 2.53 11.81 341 55 6.2 35 3-3 209.1 7.04 2.23 10.99 341 55 6.2 - 4-1 55.1 7.52 3.99 5.09 174 22 7.8 44 4-2 #DIV/0! #DIV/0! #DIV/0! #DIV/0! 174 22 7.8 - 4-3 289.2 6.07 3.35 5.49 174 22 7.8 - 5-1 80.9 7.32 2.81 10.18 517 90 5.7 30 5-2 202.8 7.29 2.89 9.68 517 90 5.7 - 5-3 58.2 7.47 3.22 6.83 517 90 5.7 - 1-1 348.20 5.81 0.00 #DIV/0! 354 65 5.4 - 1-2 1-3 2-1 2-2 24.75 24.33 130.7 269.9 -0.27 0.51 8.02 7.81 0.00 0.00 1.86 2.50 #DIV/0! #DIV/0! 10.14 9.30 354 354 10 10 65 65 56 56 5.4 5.4 0.2 0.2 11 13 43 - 119.5 5.08 1.62 10.65 #VALUE #VALUE! #VALUE #VALUE ! ! ! 452.5 8.04 2.26 9.69 74.6 7.00 2.13 11.43 114.6 5.96 1.82 13.33 61.3 5.06 1.47 18.24 54.6 6.53 3.15 7.98 10 341 56 55 0.2 6.2 39 - 0.08 341 341 174 174 174 55 55 22 22 22 6.2 6.2 7.8 7.8 7.8 28 32 63 54 - 0.18 0.20 0.75 0.02 37 29-Nov 38 2-3 3-1 3-2 3-3 4-1 4-2 4-3 obs Yield COD COD:N (mg/L) OUT NH4(mg/L) OUT (Cal) sample CH4 (mg/hr) COD (mg/L) IN NH4N (mg/L ) IN (Cal) Day NH4-N (mg/L) OUT #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! #VALUE! #VALUE ! 49.50 0.02 COD (mg/L) Consume d % COD Consume d NH4-N (mg/L) Consum ed #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 65.09 - 0 16.0 - 0 8.0 - 0 5.1 - 0 58.3 - 0 57.1 - 0 58.9 - 0 72.0 - 0 80.6 - 0 66.3 - 0 54.9 - 0 57.1 - 0 49.7 - 0 77.1 - 0 75.4 - 0 59.4 - 0 10.3 #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 54.8 65.09 15.59 56.44 56.38 - 0 0 0 0 12.0 4.6 48.6 60.0 53.1 11.0 7.9 -3.6 #REF! 54.60 - 0 0 44.6 58.3 #REF! -3.7 54.42 54.40 21.45 22.18 22.20 - 0 0 0 0 0 56.6 62.9 64.0 70.9 66.3 -2.2 -8.5 -42.6 -48.7 -44.1 341 96% #VALUE ! #VALUE ! -15 #VALUE ! #VALUE ! -150% #VALUE ! #VALUE ! #VALUE ! 306 #VALUE ! #VALUE ! #VALUE ! 90% #VALUE ! 130 #VALUE ! 75% #VALUE ! #VALUE ! 487 #VALUE ! #VALUE ! 94% #VALUE ! #VALUE ! #VALUE ! 343 341 -33 #VALUE ! -29 #VALUE ! 313 309 111 120 #VALUE ! #VALUE ! #VALUE ! #VALUE ! 97% 96% -330% #VALUE ! -290% #VALUE ! 92% 91% 64% 69% #VALUE ! 83 NO3(mg/L) NO2(mg/L) NH4-N for Cell Synthesi s 0.12 TSS NH4-N Total Organic for Nitroge n Nitroge nitrifica OUT n(mg/l) tion (mg/l) N% for Cell Synthes is Total Nitroge n IN (mg/l) Nitroge NH4-N% NH4-N% n converte d converte Conver d to to gas ted to biomass gas (mg/l) Cycle # Day sample 5-1 30-Nov 39 02-Dec 41 03-Dec 5-2 5-3 1-1 CO2 Produce d (mg) O2 (mg/hr) CH4 (mg/hr) obs Yield #VALUE #VALUE! #VALUE #VALUE ! ! ! 175.0 6.78 1.95 13.73 156.5 7.54 2.56 8.79 -11.07 -0.69 0.00 #DIV/0! COD (mg/L) IN NH4N (mg/L ) IN (Cal) COD:N COD (mg/L) OUT 517 90 5.7 - 517 517 354 90 90 65 5.7 5.7 5.4 45 46 - 1-2 -2.74 -2.04 0.00 #DIV/0! 354 65 5.4 7 1-3 31.14 -0.09 0.00 #DIV/0! 354 65 5.4 - 2-1 78.7 6.81 2.07 10.95 10 56 0.2 21 2-2 149.4 7.24 2.26 9.47 10 56 0.2 - 2-3 90.0 7.95 2.57 8.44 10 56 0.2 - 3-1 657.8 341 55 6.2 - 3-2 71.9 341 55 6.2 39 3-3 129.1 7.76 3.13 6.31 341 55 6.2 - 4-1 53.5 7.26 3.37 7.42 174 22 7.8 - 4-2 66.5 7.28 2.80 8.17 174 22 7.8 36 4-3 58.9 6.26 4.23 5.07 174 22 7.8 - 5-1 53.5 7.34 2.54 9.95 517 90 5.7 - 5-2 76.7 8.00 2.57 9.99 517 90 5.7 42 5-3 72.7 8.30 2.84 9.38 517 90 5.7 - 1-1 4.16 -0.07 0.00 #DIV/0! 354 65 5.4 - 1-2 11.38 -0.89 0.00 #DIV/0! 354 65 5.4 - 1-3 17.88 -0.12 0.00 #DIV/0! 354 65 5.4 6 #VALUE! #VALUE #VALUE ! ! 6.26 1.76 16.41 2-1 260.3 8.34 1.84 11.44 10 56 0.2 14 2-2 #DIV/0! #DIV/0! #DIV/0! #DIV/0! 10 56 0.2 - 2-3 20.7 2.83 0.98 18.03 10 56 0.2 - 3-1 1679.5 8.53 3.54 7.03 341 55 6.2 - 3-2 73.9 8.29 2.57 9.52 341 55 6.2 51 3-3 101.4 6.56 1.42 13.52 341 55 6.2 - 4-1 27.5 6.88 3.11 6.45 174 22 7.8 - 4-2 116.1 7.60 2.40 8.53 174 22 7.8 43 4-3 70.4 5.50 1.48 13.54 174 22 7.8 - 5-1 123.3 7.72 1.86 12.80 517 90 5.7 52 5-2 111.9 7.92 2.54 9.75 517 90 5.7 - 5-3 106.0 7.54 2.06 12.15 517 90 5.7 - 1-1 15.63 0.62 0.00 #DIV/0! 354 65 5.4 6 NH4(mg/L) OUT (Cal) #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! NH4-N (mg/L) OUT 1.33 2.96 #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 34.03 45.35 0.10 0.13 0.19 0.18 0.28 0.21 0.39 COD (mg/L) Consume d % COD Consume d #VALUE ! 472 471 #VALUE ! 347 #VALUE 90.30 ! 91% 88.97 91% 87.34 #VALUE #VALUE ! ! 98% #VALUE ! #VALUE #VALUE ! ! -110% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 89% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 79% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 92% #VALUE ! #VALUE #VALUE ! ! #VALUE 31.06 ! #VALUE 19.73 ! 98% #REF! #VALUE ! -11 #VALUE ! #VALUE ! #VALUE ! 302 #VALUE ! #VALUE ! 138 #VALUE ! #VALUE ! 475 #VALUE ! #VALUE ! #VALUE ! 348 NH4-N (mg/L) Consum ed NO3(mg/L) NO2(mg/L) NH4-N for Cell Synthesi s 0.12 TSS NH4-N for nitrifica tion - 0 52.0 38.3 - 0 0 0 70.9 59.4 14.3 - 0 18.3 - 0 24.6 - 0 54.3 - 0 51.4 - 0 52.0 - 0 50.3 - 0 69.1 - 0 47.4 - 0 60.0 - 0 54.9 - 0 51.4 - 0 60.6 - 0 61.7 - 0 64.0 - 0 9.1 18.1 27.9 #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 21.9 - 0 5.7 14.0 - 0 2.3 #REF! -4 -40% 56.36 - 0 58.9 -2.5 #VALUE ! #VALUE ! #VALUE ! 290 #VALUE ! #VALUE ! #VALUE ! 85% 56.33 - 0 46.3 10.0 #REF! - 0 49.1 #REF! 54.41 - 0 70.3 -15.9 54.42 - 0 69.1 -14.7 #VALUE ! #VALUE ! 131 #VALUE ! #VALUE ! 75% #REF! - 0 54.3 #REF! 22.20 - 0 57.1 -34.9 21.92 - 0 58.3 -36.4 #VALUE ! 465 #VALUE ! 90% 21.99 - 0 57.1 -35.2 89.91 - 0 68.6 21.3 - 0 71.4 18.5 - 0 72.0 #REF! - 0 6.3 #VALUE ! #VALUE ! 0.36 #VALUE ! #VALUE 348 ! #VALUE 89.94 ! #VALUE #REF! ! 98% #VALUE ! 84 Total Nitroge n OUT (mg/l) Organic Nitroge n(mg/l) N% for Cell Synthes is Total Nitroge n IN (mg/l) Nitroge NH4-N% NH4-N% n converte d converte Conver d to to gas ted to biomass gas (mg/l) Cycle # Day 42 CO2 Produce d (mg) O2 (mg/hr) 1-2 19.32 0.60 0.00 #DIV/0! 354 65 5.4 - 1-3 18.61 0.57 0.00 #DIV/0! 354 65 5.4 - 2-1 79.6 9.05 3.12 6.17 10 56 0.2 13 2-2 280.1 9.45 3.43 6.63 10 56 0.2 - 2-3 79.9 8.94 3.14 7.64 10 56 0.2 - 3-1 221.5 7.35 2.67 7.37 341 55 6.2 - 341 55 6.2 - 341 55 6.2 41 3-2 3-3 05-Dec New feed obs Yield #VALUE #VALUE! #VALUE #VALUE ! ! ! 95.0 8.94 3.19 7.77 COD COD:N (mg/L) OUT 4-1 90.2 7.02 2.53 7.85 174 22 7.8 - 4-2 54.2 6.04 1.86 11.51 174 22 7.8 33 4-3 53.8 7.96 3.36 6.50 174 22 7.8 - 5-1 60.2 7.44 3.39 6.04 517 90 5.7 46 5-2 868.9 8.62 3.12 7.05 517 90 5.7 - 5-3 497.8 8.13 2.79 6.83 517 90 5.7 - 1-1 13.90 0.71 0.00 #DIV/0! 363 58 6.2 - 1-2 48.68 0.28 0.00 #DIV/0! 363 58 6.2 5 #VALUE #VALUE! #VALUE #VALUE ! ! ! 125.2 7.19 -0.20 -103.82 363 58 6.2 - 3 50 0.1 17 1-3 2-1 44 CH4 (mg/hr) COD (mg/L) IN NH4N (mg/L ) IN (Cal) sample 2-2 229.9 6.47 2.09 10.38 3 50 0.1 - 2-3 729.0 7.34 2.58 7.31 3 50 0.1 - 3-1 100.2 6.33 2.48 9.44 373 60 6.3 62 3-2 145.9 6.06 1.89 12.30 373 60 6.3 - 3-3 166.5 5.84 1.95 12.36 373 60 6.3 - 4-1 1779.6 6.84 3.60 5.35 203 22 9.2 - 4-2 1587.8 7.03 -1.03 -17.81 203 22 9.2 38 NH4(mg/L) OUT (Cal) NH4-N (mg/L) OUT COD (mg/L) Consume d % COD Consume d #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! -3 #VALUE #VALUE ! ! #VALUE #VALUE ! ! -30% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 88% #VALUE ! #VALUE #VALUE ! ! 81% #VALUE ! #VALUE #VALUE ! ! 91% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 99% #VALUE ! #VALUE #VALUE ! ! -467% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 83% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 81% #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 300 #VALUE ! 141 #VALUE ! 471 #VALUE ! #VALUE ! #VALUE ! 358 #VALUE ! -14 #VALUE ! #VALUE ! 311 #VALUE ! #VALUE ! #VALUE ! 165 85 NH4-N (mg/L) Consum ed NO3(mg/L) NO2(mg/L) NH4-N for Cell Synthesi s 0.12 TSS NH4-N for nitrifica tion #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! - 0 2.9 - 0 6.3 - 0 50.9 - 0 60.0 - 0 63.4 - 0 52.6 - 0 61.1 - 0 66.3 - 0 53.7 - 0 57.7 - 0 58.9 - 0 58.9 - 0 63.4 - 0 54.9 - 0 4.0 - 0 2.9 - 0 1.7 - 0 54.9 - 0 56.0 - 0 48.6 - 0 61.1 - 0 60.6 - 0 62.9 - 0 50.9 - 0 48.6 Total Nitroge n OUT (mg/l) Organic Nitroge n(mg/l) N% for Cell Synthes is Total Nitroge n IN (mg/l) Nitroge NH4-N% NH4-N% n converte d converte Conver d to to gas ted to biomass gas (mg/l) Cycle # Day sample 4-3 5-1 5-2 5-3 06-Dec 46 obs Yield COD (mg/L) IN NH4N (mg/L ) IN (Cal) COD:N COD (mg/L) OUT #VALUE #VALUE! #VALUE #VALUE ! ! ! 80.1 6.49 2.11 9.11 203 22 9.2 - 561 94 6.0 67 #VALUE #VALUE! #VALUE #VALUE ! ! ! 113.8 6.08 1.92 9.82 561 94 6.0 - 561 94 6.0 - NH4(mg/L) OUT (Cal) NH4-N (mg/L) OUT COD (mg/L) Consume d % COD Consume d #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 49 45 #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 38.60 35.43 #VALUE ! 494 #VALUE #VALUE ! ! 88% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 99% #VALUE ! #VALUE #VALUE ! ! -433% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 85% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 82% #VALUE ! #VALUE #VALUE ! ! 90% #VALUE ! #VALUE #VALUE ! ! 98% 19.70 #VALUE 22.87 ! #VALUE 58.30 ! #VALUE 61.95 ! -433% 61.97 #VALUE 62.00 ! 86% 64.43 #VALUE 64.38 ! #VALUE 65.00 ! 79% 31.78 #VALUE 32.00 ! #VALUE 31.78 ! 86% 88.74 #VALUE 88.68 ! #VALUE 90.00 ! 98% #VALUE ! #VALUE #VALUE ! ! -0.59 0.00 #DIV/0! 363 58 6.2 - 1-2 #VALUE ! 28.12 0.41 0.00 #DIV/0! 363 58 6.2 - -0.57 0.00 #DIV/0! 363 58 6.2 4 1.79 1.76 9.45 3 50 0.1 - 2-2 #VALUE ! 66.8 6.42 2.13 7.53 3 50 0.1 16 2-3 72.3 5.58 2.41 6.05 3 50 0.1 - 3-1 190.0 6.52 1.87 12.49 373 60 6.3 - 3-2 88.1 7.01 2.13 11.26 373 60 6.3 57 3-3 79.7 6.61 1.68 13.85 373 60 6.3 - 4-1 74.9 6.13 2.44 7.54 203 22 9.2 - 4-2 170.4 6.39 2.47 8.12 203 22 9.2 - 4-3 68.8 6.51 1.58 13.04 203 22 9.2 37 5-1 78.6 10.05 2.35 9.36 561 94 6.0 - 5-2 63.3 7.36 2.24 10.79 561 94 6.0 56 5-3 83.1 7.22 2.12 10.49 561 94 6.0 - 1-1 1-2 20.52 24.74 -0.99 0.49 0.00 0.00 #DIV/0! #DIV/0! 363 363 58 58 6.2 6.2 8 - 1-3 20.67 -0.59 0.00 #DIV/0! 363 58 6.2 - 2-1 232.9 8.45 0.98 21.37 3 62 0.0 - 0 2-2 2-3 100.4 64.0 8.28 8.42 2.67 4.00 8.23 5.94 3 3 62 62 0.0 0.0 16 - 0 3-1 3-2 104.2 117.3 7.60 7.74 2.56 2.87 10.96 9.29 373 373 65 65 5.7 5.7 53 - 1 1 3-3 113.0 7.30 2.10 12.89 373 65 5.7 - 4-1 4-2 144.3 50.2 6.10 3.81 2.14 0.88 8.10 16.25 203 203 32 32 6.3 6.3 42 - 0 #VALUE #VALUE! #VALUE #VALUE ! ! ! 268.1 7.64 2.47 10.59 194.2 7.51 2.08 11.45 203 32 6.3 - 0 561 561 90 90 6.2 6.2 77 - 2 2 #VALUE #VALUE! #VALUE #VALUE ! ! ! 12.31 0.07 0.00 #DIV/0! 561 90 6.2 - 363 58 6.2 8 363 58 6.2 - 4-3 5-1 5-2 5-3 08-Dec CH4 (mg/hr) 35.95 2-1 07-Dec O2 (mg/hr) 1-1 1-3 45 CO2 Produce d (mg) 1-1 1-2 9.58 -0.47 0.00 #DIV/0! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 359 #VALUE ! -13 #VALUE ! #VALUE ! 316 #VALUE ! #VALUE ! #VALUE ! 166 #VALUE ! 505 #VALUE ! 355 #VALUE ! #VALUE ! 0.05 #VALUE ! 0.03 -13 #VALUE ! 0.57 320 0.62 #VALUE ! #VALUE ! 0.22 161 #VALUE ! 0.22 #VALUE ! 1.26 484 1.32 #VALUE ! 0.00 #VALUE ! #VALUE 355 ! #VALUE #VALUE ! ! 86 NH4-N (mg/L) Consum ed NO3(mg/L) NO2(mg/L) NH4-N for Cell Synthesi s 0.12 TSS NH4-N for nitrifica tion - 0 52.0 - 0 51.4 - 0 48.0 - 0 50.3 - 0 2.9 - 0 1.7 - 0 3.4 - 0 46.9 - 0 45.1 - 0 41.1 - 0 67.4 - 0 69.1 - 0 66.9 - 0 52.0 - 0 56.6 - 0 58.3 - 0 63.4 - 0 69.7 - 0 64.0 - 0 0 9.7 2.9 #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! 10.0 20.0 Total Nitroge n OUT (mg/l) Organic Nitroge n(mg/l) N% for Cell Synthes is Total Nitroge n IN (mg/l) Nitroge NH4-N% NH4-N% n converte d converte Conver d to to gas ted to biomass gas (mg/l) - 0 13.1 45.2 0 0 48.6 13.4 145 144.9 11% 163.57 18.57 29.9 70.0 0 - 0 0 50.9 54.9 11.1 7.1 140 144 140.0 10% 159.16 19.16 30.9 69.0 0 0 0 0 65.7 62.3 -1.3 2.1 125 128 124.4 127.4 8% 9% 144.43 147.73 19.43 19.73 29.9 30.4 69.2 68.7 80 85 79.8 85.0 6% 7% 91.04 98.29 11.04 13.29 34.5 41.5 64.8 58.5 165 160 163.7 158.7 11% 11% 195.07 195.25 30.07 35.25 33.4 39.2 65.2 59.4 - 0 63.4 1.6 0 0 0 0 41.1 33.7 -9.4 -1.7 - 0 47.4 -15.7 0 0 0 0 62.9 57.1 25.9 31.5 - 0 70.9 19.1 - 0 4.6 - 0 -3.4 #VALU E! #VALU E! 1-3 2-1 47 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3 09-Dec 363 58 6.2 - 3 50 0.1 22 3 50 0.1 - 3 50 0.1 - 373 60 6.3 51 373 60 6.3 - 373 60 6.3 - 203 22 9.2 - 203 22 9.2 35 #VALUE #VALUE! #VALUE #VALUE ! ! ! 63.8 6.33 1.62 11.15 203 22 9.2 - 561 94 6.0 76 #VALUE #VALUE! #VALUE #VALUE ! ! ! 98.3 6.08 1.60 13.79 561 94 6.0 - 561 94 6.0 72 69.4 6.72 1.90 8.12 #VALUE #VALUE! #VALUE #VALUE ! ! ! 56.6 6.39 1.90 11.84 127.4 6.18 2.21 10.27 #VALUE #VALUE! #VALUE #VALUE ! ! ! 324.2 6.64 1.94 9.52 71.3 5.11 1.64 11.77 1-1 23.67 -0.01 0.00 #DIV/0! 363 58 6.2 6 1-2 27.64 -1.64 0.00 #DIV/0! 363 58 6.2 - #VALUE #VALUE! #VALUE #VALUE ! ! ! 80.4 7.13 3.18 5.99 363 58 6.2 - 3 50 0.1 17 3 50 0.1 - 3 50 0.1 - 373 60 6.3 58 373 60 6.3 - 373 60 6.3 - 203 22 9.2 56 1-3 48 #VALUE #VALUE! #VALUE #VALUE ! ! ! 79.7 5.95 2.12 7.75 2-1 2-2 2-3 3-1 3-2 3-3 4-1 329.7 8.49 2.19 8.13 #VALUE #VALUE! #VALUE #VALUE ! ! ! 67.2 7.84 2.53 8.97 67.0 8.02 2.45 10.11 #VALUE #VALUE! #VALUE #VALUE ! ! ! 59.2 7.64 2.76 8.00 #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! -19 #VALUE ! #VALUE ! 322 #VALUE ! #VALUE ! #VALUE ! 168 #VALUE ! 485 #VALUE ! 489 357 #VALUE ! #VALUE ! -14 #VALUE ! #VALUE ! 315 #VALUE ! #VALUE ! 147 #VALUE #VALUE ! ! -633% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 86% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 83% #VALUE ! #VALUE #VALUE ! ! 86% #VALUE ! #VALUE #VALUE ! ! 87% #VALUE ! 98% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! -467% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 84% #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! 72% #VALUE ! 87 - 0 0.0 - 0 46.3 - 0 43.4 - 0 48.6 - 0 63.4 - 0 64.0 - 0 61.7 - 0 52.6 - 0 54.9 - 0 52.0 - 0 51.4 - 0 44.0 - 0 62.9 - 0 4.0 - 0 1.7 - 0 5.7 - 0 53.7 - 0 50.3 - 0 42.9 - 0 64.0 - 0 69.7 - 0 62.3 - 0 62.3 #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! Cycle # Day sample 4-2 4-3 5-1 10-Dec 5.04 1.18 obs Yield 17.64 #VALUE #VALUE! #VALUE #VALUE ! ! ! 87.5 8.13 2.70 8.19 COD (mg/L) IN NH4N (mg/L ) IN (Cal) COD:N COD (mg/L) OUT 203 22 9.2 - 203 22 9.2 - 561 94 6.0 - 8.39 3.26 6.91 561 94 6.0 149 5-3 76.9 8.00 2.57 9.16 561 94 6.0 81 1-1 35.74 1.41 0.00 #DIV/0! 363 58 6.2 1-2 61.95 1.89 0.00 #DIV/0! 363 58 6.2 #VALUE #VALUE! #VALUE #VALUE ! ! ! 255.7 8.97 -0.41 -60.35 363 58 6.2 3 50 0.1 2-2 420.4 9.12 3.65 6.25 3 50 0.1 2-3 74.7 9.40 3.60 6.54 3 50 0.1 3-1 81.1 8.54 3.12 9.67 373 60 6.3 3-2 335.5 7.93 2.63 12.13 373 60 6.3 373 60 6.3 203 22 9.2 203 22 9.2 203 22 9.2 561 94 6.0 561 94 6.0 561 94 6.0 363 58 6.2 #DIV/0! 363 58 6.2 #VALUE #VALUE ! ! 3.09 6.12 363 58 6.2 3 50 0.1 3 50 0.1 3-3 4-1 4-2 4-3 5-1 5-2 5-3 50 87.5 CH4 (mg/hr) 68.2 2-1 11-Dec O2 (mg/hr) 5-2 1-3 49 CO2 Produce d (mg) 1-1 #VALUE #VALUE! #VALUE #VALUE ! ! ! 81.3 7.66 2.66 8.81 78.9 5.24 0.93 23.28 #VALUE #VALUE! #VALUE #VALUE ! ! ! 369.5 8.12 3.89 5.76 55.3 8.66 2.86 9.40 #VALUE #VALUE! #VALUE #VALUE ! ! ! 36.57 0.50 0.00 #DIV/0! 1-2 40.46 0.42 1-3 0.46 2-1 #VALUE ! 89.0 2-2 88.9 8.88 3.09 7.08 2-3 89.7 9.11 3.07 5.71 3 50 0.1 3-1 67.2 8.54 3.16 8.02 373 60 6.3 3-2 64.2 8.54 3.05 9.06 373 60 6.3 3-3 70.4 8.27 2.70 10.07 373 60 6.3 4-1 74.1 7.13 2.28 8.67 203 22 9.2 4-2 35.1 4.25 1.34 14.31 203 22 9.2 4-3 59.8 7.59 2.62 8.56 203 22 9.2 5-1 83.9 8.27 2.01 11.95 561 94 6.0 5-2 103.0 8.47 2.87 8.21 561 94 6.0 5-3 77.7 8.36 3.08 7.74 561 94 6.0 8.84 0.00 NH4(mg/L) OUT (Cal) NH4-N (mg/L) OUT COD (mg/L) Consume d % COD Consume d NH4-N (mg/L) Consum ed #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE #VALUE ! ! #VALUE 412 ! #VALUE 480 ! #VALUE 363 ! #VALUE 363 ! #VALUE 363 ! #VALUE 3 ! #VALUE 3 ! #VALUE 3 ! #VALUE 373 ! #VALUE 373 ! #VALUE 373 ! #VALUE 203 ! #VALUE 203 ! #VALUE 203 ! #VALUE 561 ! #VALUE 561 ! #VALUE 561 ! 23.53 363 #VALUE ! #VALUE ! #VALUE ! 73% 100% #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! #VALUE ! 34.77 NO3(mg/L) NO2(mg/L) NH4-N for Cell Synthesi s 0.12 TSS NH4-N for nitrifica tion 100% 32.53 0 0 -10.9 43.4 10.01 3 100% 39.99 1.2585 0 47.4 -7.4 25.08 3 100% 24.92 4.1549 0 54.9 -29.9 19.19 3 100% 30.81 2.8207 0 44.0 -13.2 87.5 65 5% 98 11 21% 0.99 373 100% 58.51 0.9502 0 64.6 -6.1 73.5 72 5% 105 32 53% 0.18 373 100% 59.32 0.7744 0 70.3 -11.0 73.5 73 5% 103 30 50% 1.18 373 100% 58.32 0 0 69.1 -10.8 - 0.54 203 100% 21.46 0 0 50.9 -29.4 65 64 #VALU E! 5% #VALU E! 67 #VALU E! 2 #VALUE ! 9% 0.38 203 100% 21.62 0 0 49.1 -27.5 60 60 5% 61 1 4% 0.46 203 100% 21.54 0 0 57.7 -36.2 28.29 561 100% 65.71 0 0 61.7 4.0 130 102 7% 159 29 31% 29.11 561 100% 64.89 0 0 60.6 4.3 28.47 561 100% 65.53 0.7377 0 61.1 4.4 140 111 8% 166 26 27% 88 100% 100% 100% 100% 100% 100% 100% 66.3 - 0 9.7 - 0 6.9 - 0 7.4 - 0 56.6 - 0 52.0 - 0 53.7 - 0 69.7 - 0 73.7 - 0 69.1 - 0 54.9 - 0 50.9 - 0 52.6 - 0 53.7 - 0 64.6 - 0 57.1 0 0 31% 27.9 363 100% 0 15 4.6 25.77 100% 63.4 - 110 0 100% 62.3 0 6% 1.6278 100% 0 - 83 32.48 100% - 94.5 -0.6 100% 100% 59.4 Total Nitroge n IN (mg/l) 363 100% 58.9 0 N% for Cell Synthes is 25.82 100% 0 - Organic Nitroge n(mg/l) #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! #VALU E! 35.3 86% - Nitroge NH4-N% NH4-N% n converte d converte Conver d to to gas ted to biomass gas (mg/l) Total Nitroge n OUT (mg/l)