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Sludge reduction by mixed liquor ozonation Zheng, Xiaoyu 2018

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 SLUDGE REDUCTION BY MIXED LIQUOR OZONATION by Xiaoyu Zheng  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in The Faculty of Graduate and Postdoctoral Studies (Civil Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  February 2018 © Xiaoyu Zheng, 2018 ii Abstract  In this comparative study, a combination of mixed liquor ozone treatment and an enhanced biological phosphorus removal (EBPR) process with long SRT operations (25 d and 50 d) was tested for sludge reduction. An equivalent to about 80% sludge reduction was achieved in an EBPR system with mixed liquor ozonation and with long SRT operation.  The effluent quality was not affected with mixed liquor ozonation at SRT = 50 d. Both the control and the ozone-treated systems achieved about 90 ± 1% COD, 99 ± 1 % ammonia, 79 ± 1% inorganic nitrogen, and about 94 ± 1% ortho-phosphorus removal efficiencies at SRT = 50 d.  The mixed liquor in both systems hosted a similiar fraction of active organisms. The mixed liquor in the ozonated system exhibited a different composition of the bacteria community compared with that in the control by the Analysis of 16S rDNA. The low diversity of microbial community was observed in the ozonated reactor.  The nitrification was not affected in the ozonated system. The specific ammonium removal efficiency was comparable to that in the control. Similar fraction and diversity of nitrifiers were identified in the ozonated system and in the control. The phosphorus removal performance was not adversely affected in the ozonated system at SRT = 50 d. The maximum P-release rate and the maximum P-uptake rate in the ozonated reactor were similar to the control.  The heterotrophic decay coefficients in both reactors had no statistically siginficant difference. The maximum specific growth rate in the ozonated system (6.21 ± 0.31 d-1) was less than that in the control reactor (8.31 ± 0.55 d-1). iii The mixed liquor ozonation batch tests indicated that there was an optimum TSS/VSS concentration at which the maximum soluble COD was achieved. Carbonate/bicarbonate addition may reduce the efficiency of sludge disintegration. The solids solubilization was significantly improved under high pH conditions.  The zero sludge EBPR SBR process at lab-scale was proposed and tested. The optimum ozone dosage was determined and no inorganic matter accumulation was observed. By the batch P-release and discharge operations, the system maintained P mass balance and no P accumulation were observed. iv Lay Summary  In a municipal sewage treatment plant, the contaminants in wastewater are mainly removed by microorganisms in a bioreactor. The microorganisms, together with solid material in wastewater form sludge. The wastewater is purified by separating sludge from water by gravity settling in a secondary clarifier. Sludge contains microorganisms and many contaminants with very large volume and high percentage of water. High cost of sludge treatment and disposal is a big issue in a wastewater treatment plant.  In this dissertation, a new approach to reduce sludge production at the beginning was proposed and tested. The basic principles are: (1) a strong oxidant, ozone was intermittently introduced to a bioreactor to break some microorganisms’ cells which can be further consumed by other microorganisms as food; (2) very long sludge retention time was maintained, which allows microorganisms to decay themselves and less sludge was generated.   v Preface  I designed, constructed and operated two lab-scale MBR SBR reactors and one zero sludge lab-scale system, and conducted all the related batch experiments by myself. Dr. Eric R. Hall, my supervisor, proposed the idea of mixed liquor ozone treatment instead of recycled sludge ozonation. Mr. Timothy Ma., the technician at the Environmental Lab of UBC, helped me analyze all PO4-P, NH4-N, NOx-N, and VFA samples during the experiment.   vi Table of Contents Abstract ....................................................................................................................................... ii Lay Summary ............................................................................................................................. iv Preface......................................................................................................................................... v Table of Contents ....................................................................................................................... vi List of Tables .............................................................................................................................. x List of Figures ............................................................................................................................ xi Acknowledgements .................................................................................................................. xvi Chapter 1: Literature Review ...................................................................................................... 1 1.1 Sludge Problems in Municipal Wastewater Treatment .............................................. 1 1.2 Sludge Disintegration Technologies ........................................................................... 9 1.3 Evaluation of Sludge Reduction ............................................................................... 21 1.4 Sludge Ozone Treatment........................................................................................... 26 1.5 Sludge Ozonation Coupled to Biological Wastewater Treatment ............................ 39 1.6 Knowledge Gap ........................................................................................................ 44 1.7 Objective of the Study .............................................................................................. 47 Chapter 2: Materials and Methods ............................................................................................ 49 2.1 The Experimental Setup ............................................................................................ 49 2.2 The SBR-MBR Operational Conditions ................................................................... 60 vii 2.3 Sludge Ozonation Batch Test.................................................................................... 65 2.4 Analytic Methods ...................................................................................................... 68 2.5 Batch Test Methods .................................................................................................. 72 Chapter 3: Results of Sludge Reduction by Mixed Liquor Ozonation ..................................... 79 3.1 The Operating Conditions ......................................................................................... 79 3.2 The Inventory Changes and the Ozone Dosages ...................................................... 81 3.3 Sludge Yield Reduction ............................................................................................ 89 3.4 The Effluent Quality ................................................................................................. 96 3.5 The Characteristics of the Mixed Liquor ................................................................ 100 3.6 Summary ................................................................................................................. 106 Chapter 4: Heterotrophic and Nutrient Removal Kinetics ...................................................... 107 4.1 The Nitrogen Removal Rate ................................................................................... 107 4.2 The Phosphorus Uptake and Release Rate.............................................................. 109 4.3 The Heterotroph Kinetics ........................................................................................ 112 4.4 Summary ................................................................................................................. 120 Chapter 5: Active Biomass and Microorganisms Community ............................................... 121 5.1 Active Biomass ....................................................................................................... 121 5.2 Microscopic Observation ........................................................................................ 124 5.3 The Results of the Analysis of Genomic 16S rDNA .............................................. 127 viii 5.4 Summary ................................................................................................................. 133 Chapter 6: The Mixed Liquor Ozonation Test ........................................................................ 135 6.1 Introduction ............................................................................................................. 135 6.2 The Effect of Solids Concentration......................................................................... 137 6.3 The Effect of Sludge Source ................................................................................... 140 6.4 The Effects of Carbonates/Bicarbonates and pH on Sludge Ozonation ................. 144 6.5 Summary ................................................................................................................. 150 Chapter 7: The Zero Sludge EBPR System with Mixed Liquor Ozonation ........................... 152 7.1 The Zero Sludge EBPR System Coupled with Mixed Liquor Ozonation .............. 152 7.2 The Ozone Dosage and the Suspended Solids Inventory ....................................... 156 7.3 The Effluent Quality ............................................................................................... 158 7.4 The Batch Operation of Phosphorus Release and Discharge from the System ...... 160 7.5 Summary ................................................................................................................. 163 Chapter 8: The Conclusions .................................................................................................... 165 8.1 Introduction ............................................................................................................. 165 8.2 Overall Conclusions ................................................................................................ 165 8.3 Engineering Significance ........................................................................................ 171 8.4 Future Research ...................................................................................................... 172 Bibliography ........................................................................................................................... 173 ix Appendix A  The effluent quality for the control reactor and the ozonated reactor ............... 187 Appendix B  The identified species and their percentages of the samples from the control SBR................................................................................................................................................. 199 Appendix C  The identified species and their percentages of the samples from the ozonated  SBR ......................................................................................................................................... 238    x List of Tables Table 2.1  Influent characteristics ................................................................................................. 61 Table 2.2  Operational conditions for the control and the ozone-treated SBRs ........................... 63 Table 2.3  Types, composition and characteristics of sludge for ozone treatment ....................... 66 Table 2.4  Sample preparation protocols ...................................................................................... 69 Table 3.1  Comparison of sludge reduction by different technology ........................................... 95 Table 4.1  The nitrification specific rate ..................................................................................... 109 Table 4.2  The specific P-release rate ......................................................................................... 111 Table 4.3  The specific P-uptake rate.......................................................................................... 112 Table 4.4  The summary of the decay coefficient testing ........................................................... 113 Table 4.5  Reported decay coefficients at 20℃ .......................................................................... 115 Table 4.6  The specific growth rates of the heterotrophs for the control and the ozonated ........ 116 Table 4.7  The maximum specific biomass growth rate at 20℃ ................................................. 118 Table 4.8  The reported maximum specific growth rates of the heterotrophs at 20℃ ................ 119 Table 4.9  The Half-velocity constant (mg/L COD) at 20℃ ...................................................... 119 Table 5.1  The characteristics of flocs observed in sludge of the control and ozonated reactors 125 Table 5.2  The nitrifiers identified in sludge samples from both reactors .................................. 132 Table 7.1  Results of batch tests for phosphorus release from zero EBPR system..................... 161   xi List of Figures Figure 1.1  Physico-chemical fraction of total solids and total COD in sludge.............................. 3 Figure 1.2  Sludge treatment processes in wastewater treatment plants ......................................... 4 Figure 1.3  the relationship between sludge yield and SRT ........................................................... 6 Figure 1.4  Schematic diagram of sludge disintegration and cryptic growth ................................. 7 Figure 1.5  Combination of sludge disintegration in biological treatment unit and sludge handling unit (Category II) (Debellefontaine and Paul, 2007) .............................................................. 8 Figure 1.6  Mechanism of the direct and indirect ozonation (Gottschalk et al., 2010)................. 27 Figure 1.7  Schematic of profiles dissolved ozone in the liquid film (Foladori et al., 2010; Paul and Debellefontaine, 2007) ................................................................................................... 31 Figure 1.8  Sludge ozone treatment contactor (Park et al., 2002) ................................................. 34 Figure 1.9  Effect of ozone dosage on sludge disintegration (Foladori et al., 2010) .................... 36 Figure 2.1 The schematic diagram of the SBR-MBR system for the control ............................ 50 Figure 2.2 The schematic diagram of the SBR-MBR system combined with a mixed liquor ozone treatment reactor ......................................................................................................... 51 Figure 2.3 The diagram of the sequencing batch reactor ........................................................... 52 Figure 2.4 The photo of the SBR ............................................................................................... 53 Figure 2.5 The schematic diagram of the membrane tank ......................................................... 54 Figure 2.6 Diagram of ZeeWeed®-10 membrane module (Zenon Environmental Inc.) ........... 55 xii Figure 2.7 Diagram of the sludge ozonation treatment system .................................................. 57 Figure 2.8 Photos of the main components of the sludge ozonation treatment system      (a) the mixing tubing coil;     (b) the Venturi ozone injector and the bypass valve;     (c) the Moyno 500 series pump .................................................................................................................... 57 Figure 2.9 The curve of the positive displacement pump curve and the operating point .......... 58 Figure 2.10 The control logic of the SBR-MBR system with mixed liquor ozonation ........... 59 Figure 2.11 UBC Pilot Plant and Storage Tanks for Wastewater ............................................ 60 Figure 3.1  The DO and pH profiles for the SBRs (top: the control SBR, bottom: the ozone treated SBR) .......................................................................................................................... 80 Figure 3.2  The total mass of TSS (the inventory) in both SBRs during the 311 days of experimental operation.......................................................................................................... 82 Figure 3.3  The ozone dosage and VSS/TSS ratio for 311 days ................................................... 84 Figure 3.4  The comparison of ozone dosages by different calculations ...................................... 86 Figure 3.5  The soluble COD (SCOD), inorganic nitrogen and ortho-phosphate concentrations before and after mixed liquor ozonation ............................................................................... 88 Figure 3.6  The cumulative COD removal  and suspended solids mass balance data for the control reactor at SRT = 25 d ................................................................................................ 90 Figure 3.7  The cumulative COD removal  and suspended solids mass balance data for the ozonated system at SRT=25 d............................................................................................... 90 Figure 3.8  The observed sludge  yield comparison between the control and the ozonated reactors at SRT = 25 d. ....................................................................................................................... 91 xiii Figure 3.9  The cumulative COD removal  and suspended solids mass balance data for the control reactor at SRT = 50 d ................................................................................................ 93 Figure 3.10  The cumulative COD removal  and suspended solids mass balance data for the ozonated reactor at SRT = 50 d............................................................................................. 93 Figure 3.11  The observed yield comparison between the control and the ozonated reactors at SRT=50 d .............................................................................................................................. 94 Figure 3.12  The observed sludge yield comparison between the control and the ozonated reactors during experiment. .................................................................................................. 94 Figure 3.13  The COD concentrations for the influent and the effluent of both reactors ............. 96 Figure 3.14  The inorganic nitrogen concentrations for both reactors .......................................... 98 Figure 3.15  The ortho-phosphorus concentrations for both SBRs .............................................. 99 Figure 3.16  The SVI of mixed liquor from the control and the ozonated reactor at different SRTs............................................................................................................................................. 101 Figure 3.17  The zone settling velocity test for the mixed liquors from the control and the ozonated reactors ................................................................................................................ 102 Figure 3.18  A typical particle size distribution by volume for mixed liquor from both systems............................................................................................................................................. 104 Figure 3.19   A typical particle size distribution by number for mixed liquor samples from the  two experimental systems ................................................................................................... 105 Figure 3.20  A typical particle size distribution by surface area for mixed liquor samples from the  two experimental systems. .................................................................................................. 105 xiv Figure 4.1  The results of nitrification batch tests at SRT = 25 d ............................................... 108 Figure 4.2  The results of nitrification batch tests at SRT = 50 d ............................................... 108 Figure 4.3  The results of phosphorus release and uptake batch tests at SRT = 25 d ................. 110 Figure 4.4  The results of phosphorus release and uptake batch tests at SRT = 50 d ................. 110 Figure 4.5  The results of decay coefficient measurement ......................................................... 113 Figure 4.6  The plot of 1/S versus 1/µ for estimation of the maximum specific growth rate and half-velocity constant in the control reactor ....................................................................... 117 Figure 4.7  The plot of 1/S versus 1/µ for estimation of the maximum specific growth rate and half-velocity constant in the ozonated reactor .................................................................... 118 Figure 5.1  Total and specific cellular ATP for the control and the ozonated systems            at SRT = 25 d .......................................................................................................................... 122 Figure 5.2  Total and specific cellular ATP for the control and the ozonated systems            at SRT = 50 d .......................................................................................................................... 123 Figure 5.3  Microscopic observation of sludge flocs in two reactors (left: the control, right: the ozone treated) ...................................................................................................................... 124 Figure 5.4  protozoa observed in sludge of the control reactor .................................................. 126 Figure 5.5  protozoa observed in sludge of the ozonated reactor ............................................... 126 Figure 5.5  Affiliation and distribution of the 16S rDNA of the molecular inventory  (Up: the control;  Down: the ozonated)............................................................................................. 129 Figure 5.6  The taxonomic profile comparison at Family level .................................................. 131 xv Figure 5.7  Shannon diversity Index ........................................................................................... 133 Figure 7.2  The ozone dosage and the concentrations of TSS and VSS in the system ............... 157 Figure 7.3  TSS and VSS concentrations and VSS/TSS ratio .................................................... 157 Figure 7.4  Total COD of the influent and the effluent .............................................................. 158 Figure 7.5  Inorganic nitrogen concentrations of the influent and the effluent .......................... 159 Figure 7.6  The ortho-P concentrations of the influent and the effluent ..................................... 160 Figure 7.7  The cumulative P and P-release ............................................................................... 162 Figure 7.8  The relative P inventory in the system ..................................................................... 163    xvi Acknowledgements  I offer my enduring gratitude to the faculty, staff and my fellow students at civil engineering deparment in the UBC.  I owe particular thanks to my supervisor Dr. Eric R. Hall for his kind help, knowledgeable guidance, and insightful inspirations during my Ph.D research at UBC.  I also thank the committee members Dr. Victor Lo, Dr Don Mavinic and Dr. Madjid Mohseni for enlarging my vision of science and providing coherent answers to my questions. Special thanks are owed to my parents and my darling wife, who have supported me throughout my years of education.  1 Chapter 1:  Literature Review  1.1 Sludge Problems in Municipal Wastewater Treatment 1.1.1 Background The conventional activated sludge process that is widely used in wastewater treatment generates a large amount of excess sludge. In the USA, it has been reported that Publicly Owned Treatment Works (POTWs) generated over 8 million tons (dry weight) of sludge annually (EPA, 2006). In the EU countries, about 10 million tons (dry weight) of sewage sludge (Milieu Ltd. 2010) were produced in 2010. In China, it was estimated that 11.2 million tons dry solids were produced (Chu et al., 2009) from wastewater treatment in 2010. With increases in the population that are connected to sewage systems, the worldwide volume of sludge generated from wastewater treatment plants (WWTPs) is expected to continue to increase. The large volumes of excess sludge produced as a consequence of wastewater treatment result in significant costs for treatment, transport and disposal. Sludge treatment in WWTPs may involve thickening, stabilization, conditioning and dewatering processes, which together require about 30-80% of the total electricity used by wastewater treatment systems (EPA, 2006). The cost associated with excess sludge handling and treatment is high, constituting about 40-60% of the overall costs of wastewater treatment (Canales et al., 1994; Tchobanoglous et al., 2003; Perez-Elvira et al., 2006). The main options for sludge final disposal are agricultural use, landfilling, or incineration. However, all these disposal options are subject to stringent environmental regulations (Perez-Elvira et al., 2006). The possibility of contamination from the use of sludge limits the application 2 of sludge for agricultural use. Incineration is faced with high construction and operating costs and more stringent air emission regulations. Landfilling for sludge disposal is also constrained by the limited land availability in urban areas and strict environmental regulations.  Therefore, due to rising costs, current legal constraints, and public sensitivity towards sludge disposal, reducing and minimizing excess sludge production from wastewater treatment becomes one of most challenging tasks in the environmental engineering field.  1.1.2 Sludge Production and Composition The sludge in wastewater treatment mainly comes from the settleable solids in raw wastewater and from the heterotrophic microorganisms and small quantities of autotrophic biomass which grow on biodegradable substrate in the activated sludge process. It is estimated that specific sludge production in wastewater treatment varies widely from 35 to 85 g TS PE-1d-1 (dry total solids per population equivalent per day) (Foladori et al., 2010). The settleable solids in raw wastewater are typically of 50-60 g TS PE-1d-1 or 110-170 g total suspended solids (TSS) per cubic meter of treated wastewater (Tchobanoglous et al., 2003). The quantities of primary sludge expected can be estimated by assuming a TSS removal efficiency of 50~65% for primary treatment. The total solids content of sludge is commonly quantified with measurements of volatile suspended solids (VSS), non-volatile suspended solids and soluble organic and inorganic solids. The total chemical oxygen demand (COD) is related to the volatile solids (VS), which can be partitioned into soluble organic solids and particulate organic solids. The particulate COD of sludge is composed of autotrophic biomass (XA), heterotrophic bacteria (XH), inert particulate 3 COD (XI), endogenous residue (XP) and biodegradable particulate COD (XS), which is shown in Figure 1.1. It has been estimated that biological sludge production is 60-110 g TSS/m3 of treated wastewater during secondary treatment (Turovskiy and Mathai, 2006).  Figure 1.1  Physico-chemical fraction of total solids and total COD in sludge  1.1.3 Conventional Sludge Treatment and Disposal Processes Because excess sludge contains about 98~99% water (Tchobanoglous et al., 2003), it is necessary to reduce the water content before it is transferred out of a wastewater treatment plant for final disposal. The typical sludge treatment processes in a wastewater treatment plant include preliminary operations (e.g. storage, grinding, blending and/or degritting), thickening (e.g. settling, flotation, centrifugation, etc), stabilization (e.g. anaerobic digestion, aerobic digestion and/or lime stabilization), conditioning (chemical addition and coagulation), and dewatering (e.g. centrifuge, belt press, drying beds, etc.); and (6) disposal, further processing or land application. The general sludge processing flow diagram is shown in Figure 1.2. 4  Figure 1.2  Sludge treatment processes in wastewater treatment plants The sludge final disposal options are variable and influenced by many factors such as climatic conditions, land usage and cost, agriculture fertilizer needs, transport costs, capacity of treatment facilities, local regulations, economy and other conditions (Foladori et al., 2010). The normal sludge disposal options include: (1) on-site storage; (2) ponds or lagoons; (3) landfilling; (4) agriculture/land application; (5) composting; (6) incineration; and (7) industrial reuse. Traditionally, land application and landfilling are the cheapest options for sludge disposal. However, with the continuous increase in sludge production, limited land availability and more stringent regulations, the cost for sludge disposal greatly increases. A mean cost of 470 ± 280 €/t TS was estimated (Paul et al., 2006) for sludge treatment, transportation and disposal in European countries.  1.1.4 Emerging Sludge Reduction Strategies The ideal solution for sludge reduction is to minimize excess sludge production from wastewater treatment processes, rather than treating and disposing of the sludge after generation (Chen et al., 2003). Since the 1990’s, many studies have been conducted to develop technologies for direct on-site sludge reduction. The fundamental approaches available to directly reduce the generation 5 of sludge are generally either: (1) yield reduction, and (2) in-line sludge disintegration by physical, chemical or thermal methods. The yield reduction approach involves the introduction of additional stages in wastewater treatment processes with a lower cellular yield coefficient (Perez-Elvira et al., 2006). The options include: (1) cell lysis and cryptic growth; (2) uncoupled metabolism; (3) endogenous metabolism; and (4) microbial predation. The process of uncoupled metabolism entails the uncoupling of energy intended for anabolism and reducing the growth yield by adding chemical uncoupling compounds. However, most of chemical uncouplers tested are xenobiotic compounds and potentially harmful to the environment (Wei et al., 2003) and thus, their application is limited. The microbial predation process introduces protozoa and metazoa to graze on the bacteria in the sludge. Although the worms introduced may reduce  sludge generation, practical application is still not feasible due to the difficulty in controlling operational parameters such as worm growth (Wei et al., 2003; Perez-Elvira et al., 2006). Endogenous metabolism or maintenance metabolism reduces sludge production by increasing the solids retention time (SRT) or decreasing the food-to-microorganism ratio. A membrane bioreactor (MBR) treatment process can be operated successfully at relatively short hydraulic retention times (HRT) and long solids retention times (SRT). It has been reported (Wagner and Rosenwinkel, 2000) that minimum sludge production was observed in a pilot scale submerged MBR with a capacity of 220 L operating for one year without sludge discharge.  If the formula C10H19O3N for domestic wastewater and the formula C5H7NO2 for microbial biomass are assumed, the biomass yield versus SRT can be calculated according to the 6 stoichiometry and bacterial energetics principal proposed by McCarty and Bruce (2012). Figure 1.3 shows the relationship between sludge yield and SRT from the model calculation when biodegradable fraction of biomass fd=0.8 and death rate of microorganisms b=0.1 d-1 are assumed. The sludge yield curve becomes flat at longer SRTs, and sludge production will be reduced if long SRT operation is adopted. The most widely investigated emerging sludge reduction approach is cell lysis and cryptic growth (Figure 1.4). In this approach, microbial organisms can take up lysate, the contents of lysed cells, as their carbon energy substrate or nutrient sources. The biomass grows on lysate, which is different from the original substrate and therefore, the growth is termed as cryptic growth (Mason, 1986). The rate-limiting step for lysis-cryptic growth is the lysis stage. To facilitate cell lysis, sludge disintegration processes have been developed in recent years and these have become the pivot to achieve sludge reduction and minimization.  Figure 1.3  the relationship between sludge yield and SRT  0.10.20.30.40.50.60.70.80 25 50 75 100Yield  SRT (d) 7  Figure 1.4  Schematic diagram of sludge disintegration and cryptic growth  1.1.5 Sludge Disintegration in Wastewater Treatment Plant Sludge disintegration is a cell lysis treatment of sewage sludge using external forces such as those induced by mechanical, thermal, chemical, or electrical processes to change the floc structure and/or to disrupt the microbial cell wall, to enhance the solubility of sludge particulates and to improve subsequent biological degradation (Bougrier et al., 2006; Muller, 2001; Winter, 2002).  Sludge disintegration can be coupled to biological wastewater treatment (Category I) or a sludge handling process (Category II) in a wastewater treatment plant. Figure 1.5 shows the possible combinations of sludge disintegration and wastewater treatment as described in (Debellefontaine and Paul, 2007). 8  Figure 1.5  Combination of sludge disintegration in biological treatment unit and sludge handling unit (Category II) (Debellefontaine and Paul, 2007) To combine sludge disintegration with a biological wastewater treatment process, a fraction of the recycled sludge, the waste sludge or the mixed liquor can be treated to promote the solubilization and biodegradation of sludge. The treated sludge is returned into the bioreactor, where the released biodegradable COD is oxidized by heterotrophic bacteria or utilised as an additional carbon source to enhance nitrogen removal. Although additional lysates promote a further growth of biomass, the overall observed growth yield decreases and sludge production can be directly reduced. The retention time of active cells in biological treatment should not be affected due to sludge disintegration so that the performance of the wastewater treatment system does not deteriorate. Sludge disintegration can be used as pretreatment for a sludge handling process, promoting hydrolysis in sludge digestion. The aerobic or anaerobic sludge digestion processes are mainly 9 limited by cell lysis and particulate hydrolysis (Lafitte-Trouque & Forster, 2002). By applying sludge disintegration pretreatment, the initial stage of hydrolysis can be improved and the volume of the digester and the residence time required for digestion can be minimized. In addition, biogas production and energy recovery can be increased and sludge quantity for disposal can be reduced.   1.2 Sludge Disintegration Technologies A variety of sludge disintegration techniques can be used for sludge treatment, including mechanical, ultrasonic, thermal, chemical, electrical, and other treatments. A hybrid treatment, which combines two disintegration processes, may be used for cell lysis.  1.2.1 Mechanical Disintegration In mechanical disintegration, energy is supplied to generate pressure, or rotational/translation movement on the solids which results in tensions and deformations of the sludge flocs and bacterial cells (Muller, 2001). Because of the strength of the cell wall, the cell of a microorganism can resist considerable external tension or stress. At lower energy inputs, only floc disaggregation is observed. Bacterial cell disruption requires high energy input. The main mechanical sludge disintegration techniques include stirred ball mills; high pressure homogenizers, high pressure jets and collision, nozzle-cavitation and ultrasonic disintegration. Stirred ball mills (SBM) consist of a cylindrical grinding chamber with a rotating central crankshaft. The volume of the chamber is up to 1 m3, which is filled with grinding beads with diameter between 0.2-2 mm (Baier and Schmidheiny, 1997). The beads are made of either steel 10 or ceramic materials. During sludge disintegration, the rotating crankshaft with blades forces the beads into a rotational movement and the flocs and cells are disintegrated between the beads by shear and pressure forces (Muller, 2000). After sludge disintegration, the beads are held back by a sieve while the suspension flows through the grinding chamber (Winter, 2002). However, the potential degree of disintegration by using SBM is limited. The operating problems, such as clogging of the spheres, separation sieves and wear of the machine components, are other drawbacks. High pressure homogenizers (HPH) consist of a multistep high pressure pump and an adjustable homogenisation valve. The pump compresses the sludge to pressures up to several hundred bar. By passing through the homogenisation valve, the sludge velocity increases up to 300 m/s. The velocity increase causes a rapid drop of pressure to below vapor pressure and cavitation bubbles implode, resulting in temperatures of several hundred degrees Celsius and pressure peaks of 500 × 105pa locally (Lehne et al., 2001; Muller, 2000). The high temperature and the pressure gradients are the main causes of the floc disaggregation and cell rupture (Strunkmann et al., 2006). The effect of HPH treatment is influenced by homogenization pressure, the number of homogenization cycles applied and the suspended solids concentrations in the sludge.  It has been reported (Strunkmann et al., 2006) that a combination of a membrane bioreactor (MBR) and mechanical disintegration can achieve up to 70% excess sludge reduction and no obvious adverse effect was observed on the wastewater treatment COD removal efficiency. However, nitrogen removal deteriorated as a result of the sludge disintegration to some degree. The higher sludge concentration (45.3 g/kg) in an MBR results in lower specific energy consumption by the mechanical disintegration; and the thickened sludge undergoes more 11 extensive disintegration by a high pressure homogenizer, a stirred ball mill or an ultrasonic homogenizer (Lehne et al., 2001).  1.2.2 Ultrasonication Ultrasound is cyclic sound pressure wave with a frequency greater than 20 kHz. By generating compressions and rarefactions, ultrasound in water can induce cavitation bubbles, which grow in successive cycles and reach an unstable size before they collapse violently, resulting in: (1) hydrodynamic shear forces, and (2) locally high temperatures of around 5000°C and pressure of 500 atmospheres in a few microseconds, which form highly reactive radicals (OH·, HO2·, H·) and hydrogen peroxide (Khanal et al., 2007; Pilli, Bhunia et al., 2011). Since the 1990’s, ultrasound has been proposed for sludge reduction (Chiu et al., 1997; Tiehm et al., 2001). Both hydrodynamic shear forces and highly reactive radicals can destroy flocs and bacterial cells in sludge and release the intracellular matter.  The performance of ultrasonic sludge disintegration is determined by three factors: (1) applied energy; (2) ultrasound frequency; (3) sludge properties. The main parameters characterizing the energy applied are: power intensity (power per unit area of the sound-emitting surface, W/cm2), power density (power per unit volume of sludge treated, W/L), specific energy or ultrasonic dose (energy per unit volume of sludge treated, J/L), and specific energy (energy per unit of TS in sludge treated, kJ/kg TS). In the literature, the most widely used parameter is the specific energy (Es) which is calculated as, 𝐸𝑠 = 𝑃∙𝑡𝑉∙𝑥  (1-1) 12 Where, P = applied power (W); t = treatment time (s); V = treated volume (L) and x = sludge concentration (g TS/L). Generally, an increase of Es results in an increase in the extent of sludge disintegration. Low Es levels mainly cause floc size reduction without significantly damaging bacterial cells. Higher Es levels are required to cause cell lysis. A linear relationship between COD solubilization and the logarithm of Es has been reported (Andreottola and Foladori, 2006; Bougrier et al., 2005; Zhang et al., 2007). At Es < 32,000 kJ/kg TSS, the main effect of ultrasonic disintegration is floc size reduction and the concentration of intact and dead cells released to the bulk liquid increases significantly, up to 10 times the concentration before treatment. At Es > 32,000 kJ/kg TSS, the number of intact bacterial cells starts to decrease due to the progressive rupture of cells. At around 93,000 kJ/kg TSS, COD solubilization was 30.1% and solids reduction was 23.9%, while almost 95.5% biomass inactivation was observed by the oxygen uptake rate (OUR) test (Zhang et al., 2007). Power intensity or power density also affects sludge disintegration. At the same Es, a high power input with a short treatment time results in high degree of COD solubilization (Show et al., 2007). Ultrasound frequency is another important parameter that affects the performance of sludge ultrasonication. Higher ultrasound frequencies favour hydroxyl radical generation and chemical reaction, while lower ultrasound frequencies produce stronger cavitation effects (Zhang et al., 2008). At lower ultrasound frequencies, hydrodynamic shear forces produced by ultrasonic cavitation are primarily responsible for sludge disruption (Tiehm et al., 2001). Sludge disintegration tests commonly have been conducted in the lower frequency range of 20 kHz (Akin et al., 2006; Bougrier et al., 2005; Wang et al., 2005). However, a recent study (Gallipoli 13 and Braguglia, 2012) reported that high-frequency (200 kHz) treatment permitted a higher degree of disintegration compared to that with a low frequency of 20 kHz sonication for specific energy input > 30,000 kJ/kg TS. Sludge properties such as TS concentration affect the sludge disintegration process. At a higher TS concentration in the bulk liquid, a higher number of cavitation sites occurs and the probability that the solids come into contact with exploding cavitation bubbles increases. At the same Es applied, the COD solubilization is lower for a concentration of 10.5 g TS/L than that at 34.4 g TS/L (Neis et al., 2000). However, there is a limit in TS concentration at around 30 g TS/L, beyond which efficiency decreases (Mao et al., 2004; Show et al., 2007) because the liquid-solid sludge system must contain a sufficient quantity of liquid to allow the formation of micro-bubbles during cavitation.  The major advantages of ultrasound treatment are reliability of operation, limited odor generation, minimal clogging problems, compact design and easy retrofit within an existing system, with the additional potential to control filamentous bulking and foam generation. The major limitations are high capital and operating costs of ultrasound units with high energy consumption. Long-term performance data of full-scale ultrasound treatment are also limited (Hogan et al., 2004).  1.2.3 Thermal Treatment  Thermal treatment is an effective method to disintegrate sludge that is achieved by applying high temperatures. Thermal treatment in the temperature range from 60 to 180 °C can break down the sludge structure, disaggregate biological flocs, disrupt the chemical bonds of the cell wall and 14 release intracellular constituents (Appels et al., 2008). It has been observed (Bougrier et al., 2006; Camacho et al., 2005; Dohanyos et al., 2004) that COD solubilization increased in approximately a linear relationship with temperature and that contact time had little impact, especially at higher temperatures. Conventional thermal treatment is a relatively simple method and heat is transferred from the heating device to the medium. The thermal energy is usually provided by heat exchangers or by applying steam directly to the sludge. The performance of thermal treatment depends on thermal conductivity, temperature gradients and convection. The major drawbacks are a high energy requirement, fouling of the heat exchangers, odor problems and high operating costs (Perez-Elvira et al., 2006). In recent years, the use of microwaves for heating has been proposed as an innovative technique to treat sludge (Eskicioglu et al., 2008; Hong et al., 2006; Park et al., 2006). Compared with conventional heating, microwave irradiation reduces the reaction time and energy requirement due to lower thermal losses in heat transfer. However, the effect of microwaves on microorganisms is not yet fully understood and microwave technology has not yet been successfully applied at full-scale.  1.2.4 Chemical Treatment  Chemical sludge treatment causes the hydrolysis of cell walls or membranes to release and solubilize organic matter contained within the microbial cells, by applying strong acids, alkali and oxidants such as ozone and peroxides. 15 The alkali/acidic treatment methods are relatively simple and effective. About 50 ~ 60% solubilization of sludge can be achieved by adding 0.5 - 1.0 g sulfuric acid/g TSS for 30 seconds at room temperature (Woodard and Wukasch, 1994). However, such high dosages of acid seem impractical for sludge disintegration. Alkali treatment is more effective than acidic treatment. It has been reported (Tyagi and Lo, 2011) that a 45 meq/L NaOH dose can achieve 27.7, 31.4 and 38.3% COD solubilization at 25, 35 and 55°C, respectively, with 4 h reaction time. Over 50% of the VSS was solubilized with 0.2 mol/L NaOH with 8 h reaction time (Li et al., 2009). The dosage of 0.08 g NaOH/g TSS achieved 83% COD solubilization (Lin et al., 2009). However, the major drawbacks are odor generation, corrosion and fouling of equipment, and the subsequent neutralization requirement. Oxidants can disintegrate the extracellular polymeric substances (EPS) and rupture the cell walls, thus, the intracellular material can be released, which leads to a soluble COD increase. Several oxidants have been reported for application in sludge disintegration, including ozone (Chu et al., 2009; Lin et al., 2001; Yeom et al., 2002), Fenton peroxidation (Dewil et al., 2007), chlorination (Liu, 2003; Saby et al., 2002) and peracetic acid (PAA) (Shang and Hou, 2009). Among these oxidants, ozone is the most widely used. Ozone is a strong chemical oxidant that is commonly used in water and wastewater treatment.  In recent decades, ozone has been reported to be used to treat excess sludge and a high degree of disintegration was achieved (Muller, 2000). Compared with other sludge disintegration technologies, ozone treatment has many advantages: (1) ozone can be generated by electricity on-site and no other chemicals are required; (2) the pH value need not be adjusted during reaction and no additional salts are generated; (3) a high degree of sludge disintegration can be achieved; (4) sludge settling and dewatering conditions can be improved significantly and 16 bulking and foaming can be reduced effectively. However, the main drawback for ozonation is its high energy consumption. It is estimated that 9 - 15 kwh/kg O3 is required for ozone production (Foladori et al., 2010), in addition to 2.5 kwh/kg O3 for transfer to the sludge (Tyagi and Lo, 2011), which costs about 1.8 US dollars (He et al., 2006). Sludge ozonation will be described in detail in section 1.4.  1.2.5 Other Treatments  Pulsed electric field treatment is a recently proposed method which uses pulsed electric currents in the sludge stream to cause shock waves that induce cell lysis. The sludge is treated by a high voltage of up to 10 kV with pulse periods of only 10 milliseconds, to induce the sudden disruption of sludge flocs and microbial cells to release soluble organic material (Muller, 2001). It was reported (Kopplow et al., 2004) that 20% sludge solubilization was achieved at a specific energy input of 8,000 kJ/kg DS. The major drawbacks of this method are a lack of research data, the erosion of the electrodes and the high-energy input requirement (Tyagi and Lo, 2011). Hydrodynamic cavitation can also be used for sludge disintegration treatment. Unlike sonication, cavitation can be induced on the basis of a hydrodynamic principle of the Venturi effect, in which the bubbles are generated when the pressure in the constricted region (Venturi throat) falls below the vapor pressure of the liquid (Kim et al., 2008).  An electron beam can also be applied for sludge disintegration. The irradiation dose of the electron beam varies from 0.5 to 10 kGy. It was observed (Shin and Kang, 2003) that 30-52% sludge COD solubilization could be achieved with electron beam irradiation for 24 h reaction time. 17 Gamma irradiation is another method for sludge disintegration. Gamma irradiation can be generated directly by ionizing particles or indirectly from a radionuclide source. Gamma rays can inactivate microorganisms and decompose various organic compounds. It was reported (Lafitte-Trouque and Forster, 2002; Yuan et al., 2008) that the γ-irradiation treatment of sludge increased the soluble COD significantly. However, the high energy consumption and a lack of research data are major drawbacks of this technique.  1.2.6 Hybrid Treatment Hybrid sludge disintegration methods are the combination of a physical or mechanical method with a chemical method. These tend to achieve higher efficacy compared with a single technique due to the synergistic effect of the combined treatment. The common hybrid disintegration methods include: (1) chemically enhanced thermal treatment, (2) alkali-ultrasonic treatment, (3) ultrasonication-ozonation, and (4) other methods. Thermal-chemical treatment involves thermal pretreatment followed by the addition of an acid or base to solubilize the sludge and avoid the necessity of high temperatures in the pretreatment stage. Thermal-chemical treatment was observed to accelerate COD solubilization and enhance the efficacy of anaerobic digestion (Andreottola and Foladori, 2006; Penaud et al., 2000; Tanaka et al., 1997). The full scale installation of thermal-chemical treatment was reported to achieve about 40% solubilization of particulate organic matter at a temperature of 140°C at about 3.5 bars for 30 - 40 min (Odegaard, 2004). The advantages of this method are an enhancement of sludge reduction with low chemical consumption and high phosphate release. However, additional costs due to the consumption of reagents and the energy required for heating, odor 18 generation and corrosion control, together with high operation and maintenance costs are the main disadvantages of the method. Alkali-ultrasonic treatment combines alkaline treatment and sonication. It is assumed that the use of an alkali such as NaOH may weaken the cell walls making them more susceptible to the sonication lysing process. It was found (Chiu et al., 1997) that a high rate of sludge hydrolysis could be achieved after sludge pretreatment with simultaneous addition of NaOH and 20 kHz ultrasonication. Several researchers observed that more than 50% VS solubilization can be achieved after combined ultrasonication- alkali pretreatment (Liu et al., 2008; Jin et al., 2009). Ultrasonication-ozonation is another hybrid sludge treatment which couples ozonation with the efficient ultrasonic cavitation. The dispersion of activated sludge flocs by ultrasonic cavitation increases the specific surface of aggregates and the susceptibility to the oxidants in the medium. The ultrasonic waves may also enhance the mass transfer of ozone. The cavitation bubbles can also improve the ozone reaction and free radical generation (Jyoti and Pandit, 2004). It was reported (Xu et al., 2010) that the soluble COD could be increased from 83 to 2,483 mg/L after 60 min ozone treatment followed by 60 min ultrasound treatment, while the soluble COD increased to 3,040 mg/L after 60 min of ultrasound/O3 treatment. This indicated that ultrasound/O3 induced a synergistic effect on COD solubilization. The hybrid sludge disintegration methods may be promising alternatives to enhance sludge solubilization while neutralizing the associated drawbacks of a single technique at the same time. However, currently research on the application of hybrid pretreatment is in its early stage and more studies and data are needed.  19 1.2.7 Summary Each sludge disintegration method has advantages and drawbacks. Most of the available sludge disintegration methods are either energy-intensive and/or they require high capital and operating costs. Table 1.1 presents a comparative evaluation of various sludge disintegration treatment techniques. Mechanical methods require high capital and operating costs, and their sludge reduction potential is limited. Ultrasonication and thermal treatment have been shown to be effective methods to disintegrate sludge; however, the energy consumption is very high. Chemical treatment methods are more energy economical and can achieve a high degree of sludge solubilization, but they can cause corrosion problems for equipment components and may require post-neutralization, which leads to high operational and maintenance costs. Hybrid treatments, which combine different physical-chemical-mechanical technologies may be superior to individual methods due to the benefits of possible synergistic effects; however, little research has been reported on these to date.  20 Table 1.1  Evaluation of various pretreatment methods Reference (Paola et al., 2010; Muller, 2001; Muller et al., 2004; Tyagi and Lo, 2011) Sludge disintegration method Sludge reduction Energy requirement Capital Cost O&M cost Operational problems Technology maturity Physical treatment Stirred ball mills 12-14%  (VS) high high high Clogging, spheres separation, machine wear Lab/pilot/full scale High pressure homogenisers 20-24% (TSS) medium medium medium-high Clogging, high tension and erosion in the pump and valve Lab/pilot/full scale Ultrasonication 25-90%(TSS) high high high Erosion in the sonotrode  Lab/pilot/full scale Electric pulse  -- Medium-high high high Erosion in the electrode, low research and development Lab Gamma or electron beam irradiation -- high high high No significant improvements Lab Thermal treatment Conventional Thermal  <55% (TSS) high high high or low Fouling of the heat exchangers, possible bad odour Lab/pilot/full scale Microwave  -- medium medium medium-high possible bad odour Lab scale Chemical treatment Acid-Alkali High (depending on dosage) low low medium Corrosion and fouling, bad odour Lab/pilot scale Ozonation High (depending on dosage) high high high foaming Lab/pilot/full scale Hybrid treatment Thermo-chemical High medium medium high Corrosion Lab/pilot/full scale Mechanical-chemical High low medium medium Possible corrosion or foaming Lab/pilot Thermo-mechanical High high high high Corrosion, possible bad odour Lab/pilot   21 1.3 Evaluation of Sludge Reduction The efficiency of sludge reduction can be assessed by estimating the observed sludge yield (Yobs) during experiments at pilot-scale or at lab-scale to simulate full-scale plant configurations. However, the results from lab and pilot plants generally require long monitoring times and high cost. Therefore, quick tests are needed to evaluate the efficiency of sludge reduction techniques. The available rapid tests include TSS and COD solubilization, inactivation of bacteria, and sludge biodegradability. In addition, when pilot plant tests are used, the operational parameters such as sludge retention time, observed sludge yield and treatment flow rate or frequency need to be considered and calculated.  1.3.1 The Extent of Sludge Solubilization The primary objective of sludge disintegration is to solubilize the suspended solids and to improve biodegradability instead of wasting and treating them in an additional sludge handling unit. The evaluation of COD and TSS solubilization can give information on the transformation of particulate COD into soluble COD. The extent of sludge disintegration (%), ε, is defined as the ratio of the solubilized solids mass to the mass of total suspended solids (TSS) disintegrated as described in the following expression,  𝑇𝑆𝑆 𝑑𝑖𝑠𝑖𝑛𝑡𝑒𝑔𝑟𝑎𝑡𝑖𝑜𝑛(%), 𝜀 = (𝑇𝑆𝑆0−𝑇𝑆𝑆𝑡)𝑇𝑆𝑆0× 100%   (1-2) Where, ε = the extent of TSS solubilization, %; TSS0 = the concentration of total suspended solids in the sludge before disintegration treatment, mg/L; TSSt = the concentration of total suspended solids in the sludge after treatment, mg/L. 22 Some researchers ( Bougrier et al., 2005; Cui and Jahng, 2006; El-Hadj et al., 2007; Yan et al., 2009) also have used the degree of COD solubilization, which is calculated as the following equation, 𝐶𝑂𝐷 𝑠𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑠𝑎𝑡𝑖𝑜𝑛 = 𝑆𝐶𝑂𝐷(%) = 𝑆𝐶𝑂𝐷𝑡−𝑆𝐶𝑂𝐷0𝐶𝑂𝐷0−𝑆𝐶𝑂𝐷0× 100%  (1-3) Where, SCOD0 = the concentration of soluble COD in the sludge before disintegration, mg/L; SCODt = the concentration of soluble COD in the sludge after disintegration, mg/L; COD0= the concentration of total COD in sludge before disintegration, mg/L. Some authors (Saktaywin et al., 2005) took into account the particulate COD and defined the degree of COD solubilization as, 𝑆𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑠𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑝𝑎𝑟𝑡𝑖𝑐𝑢𝑙𝑎𝑡𝑒 𝐶𝑂𝐷(%) = 𝑃𝐶𝑂𝐷(%) = 𝑃𝐶𝑂𝐷0−𝑃𝐶𝑂𝐷𝑡𝑃𝐶𝑂𝐷0× 100%  (1-4) Where, PCOD0 = the concentration of particulate COD in the sludge before disintegration, mg/L; PCODt = the concentration of particulate COD in the sludge after treatment, mg/L. The extent of COD solubilization defined in Equation (1-1) assumes that the total COD concentration remains the same before and after treatment. However, if some disintegration techniques such as ozonation are used, mineralization of COD also occurs. In this case, the expressions in Equation (1-2) and (1-3) are more valid. In addition, to calculate a suspended solids mass balance in a zero sludge system, the extent of TSS disintegration seems to be more useful than the COD solubilization ratio.  The extents of COD and TSS solubilization are approximate indicators of sludge disintegration which are widely reported in the literature, especially in the case of sludge reduction in wastewater treatment process (Category I in Figure 1.5).  23 1.3.2 Degree of Disintegration The degree of disintegration is a parameter to evaluate physical treatments for sludge disintegration. The degree of disintegration is determined on the basis of COD solubilization (Foladori et al., 2010). The degree of disintegration (%), DDCOD, is defined as (Muller, 2000), 𝐷𝐷𝐶𝑂𝐷(%) = 𝑆𝐶𝑂𝐷𝑡−𝑆𝐶𝑂𝐷0𝑆𝐶𝑂𝐷𝑁𝑎𝑂𝐻−𝑆𝐶𝑂𝐷0 × 100%   (1-5) Where, 𝑆𝐶𝑂𝐷0 = concentration of soluble COD in the untreated sludge, mg/L; 𝑆𝐶𝑂𝐷𝑡 = concentration of soluble COD in the sludge after treatment, mg/L; 𝑆𝐶𝑂𝐷𝑁𝑎𝑂𝐻 = maximum COD concentration that can be solubilized, which is determined by an alkaline process using 0.5 mol/L or 1.0 mol/L NaOH for 22-24 hours at room temperature (Bougrier et al., 2005; Gonze et al., 2003; Tiehm et al., 2001), mg/L. The concentrations of soluble COD in sludge are determined after 0.45 μm filtration or in the supernatant after centrifugation. Some researchers have also used H2SO4 digestion, rather than NaOH, to measure the maximum soluble COD (Braguglia et al., 2006).  The degree of disintegration can also be evaluated by using oxygen consumption (Kopp et al., 1997). The degree of disintegration (%), DDO2, is defined as, 𝐷𝐷𝑂2(%) = �1 − 𝑂𝐶𝑡𝑂𝐶0� × 100%  (1-6) Where, 𝑂𝐶0 is the oxygen consumption of the untreated sludge, g; and 𝑂𝐶𝑡 is the oxygen consumption of the treated sludge, g.  However, the oxygen consumption is measured over a short period and the respiration is not considered. To investigate bacterial damage and the release of biodegradable matter, the 24 measurement of oxygen uptake rate (OUR) was proposed (Camacho et al., 2002) to estimate heterotrophic biomass inactivation (I), 𝐼(%) = �1 − 𝑂𝑈𝑅𝑡𝑂𝑈𝑅𝑚𝑎𝑥� × 100%    (1-7) Where, 𝑂𝑈𝑅𝑚𝑎𝑥 is the maximum specific OUR before treatment, mg O2/L/min; and 𝑂𝑈𝑅𝑡 is the OUR after treatment, mg O2/L/min.  1.3.3 The Biodegradable COD Solubilization The biodegradability of the organic compounds released by sludge disintegration is a reliable and representative parameter to evaluate the sludge reduction potential. The biodegradability can be evaluated by respirometric tests (Paul et al., 2006). The integration of the respirogram gives the net amount of oxygen (ΔO2) consumed during the biodegradation over a period of time. Then the biodegradable COD can be calculated from the net amount of oxygen (ΔO2) by, 𝑆𝑠 = ∆𝑂21−𝑌 × 𝑉0+𝑉𝑡𝑉𝑡    (1-8) Where, 𝑆𝑠 = the concentration of biodegradable COD, mg/L; 𝑌 = heterotrophic yield value, 0.67 g COD/g COD; 𝑉0 = the volume of activated sludge in the respirometer reactor, L; 𝑉𝑡 = filtered sludge volume after disintegration treatment, L. Another rapid physical-chemical method (Mamais et al., 1993) to determine readily biodegradable soluble COD can be used. The total soluble COD (SCOD) is determined by membrane filtration of a sample that has been flocculated (Zn(OH)2 at pH = 10.5). The non-readily biodegradable soluble COD (Si) is determined by performing a soluble COD analysis on 25 the effluent of a 24 hr fill and draw activated sludge system (SRT > 3days) fed with the wastewater of interest. Then the readily biodegradable soluble COD (Ss) can be calculated by, 𝑆𝑠 = 𝑆𝐶𝑂𝐷 − 𝑆𝑖  (1-9) Where, 𝑆𝑠 = readily biodegradable soluble COD; 𝑆𝐶𝑂𝐷 = total soluble COD; and 𝑆𝑖 = non-readily biodegradable soluble COD.  1.3.4 Bacterial Inactivation In sludge disintegration treatment, microbial cells are disrupted and the intracellular matter is released. To investigate the efficiency of a disintegration treatment for the lysis of cells, both conventional and innovative methods can be used. Microscopic observation is a conventional approach that is used to assess the disaggregation or dispersion of flocs after sludge disintegration treatment. This method can give a qualitative description of sludge disintegration treatment by examination of the microscopic images. The Oxygen Uptake Rate (OUR), which is calculated by measuring the dissolved oxygen (DO) concentration over time through respirometry, is another way to estimate the activity of biomass. In addition, the specific contaminant removal rate such as nitrification rate and phosphorus uptake and relase rate, can also be used to assess the activity of certain microorganisms.  Advanced in situ techniques can be used for rapid quantification of bacterial populations and their physiological states in sludge. A recent study (Foladori et al., 2010) showed that coupling fluorescent molecular probes followed by flow cytometry (FCM) can rapidly and accurately quantify the intact, permeabilised or disrupted bacterial cells in the sludge.  26 Adenosine triphosphate (ATP) is the primary energy carrier for microorganisms. The measurement of celluar ATP concentration in a sludge sample provides a direct measurement of biological acitivity. The ATP is quantified by using a luminometer.  1.4 Sludge Ozone Treatment Among current sludge disintegration methods, ozonation is widely used because it requires no chemicals and it offers high performance for sludge solubilization and biodegradation. Ozonation could be one of the most promising technologies for sludge disintegration if it is combined with other techniques such as mechanical treatment to improve treatment efficiency and reduce energy consumption. The performance of sludge ozonation is dependent on the chemical reaction mechanism, ozone mass transfer, reactor configuration and ozone dosage.  1.4.1 Chemical Reaction Mechanism Ozone can react with substances in two ways: (1) direct reaction with a compound; and (2) via hydroxyl radicals which can indirectly react with substances.  Figure 1.6 shows the indirect and direct pathways and their interaction.  The indirect reactions can be divided into three steps: the initiation step, the radical chain and the termination steps.  At the first step, the ozone decays and is accelerated by initiators such as OH¯, to form superoxide anion (O2°¯) and hydroperoxyl radical (HO2°), which is in an acid-base equilibrium. 𝑂3 + 𝑂𝐻−  → 𝑂2°− + 𝐻𝑂2°    (1-10) 27 𝐻𝑂2° ↔ 𝑂2°− + 𝐻+  (1-11) The ozone then reacts with the superoxide anion radical (O2°¯) to form the ozonide anion radical (O2°¯), which decomposes into a hydroxyl radical (OH°).  𝑂3 + 𝑂2°−  → 𝑂3°− + 𝑂2  (1-12) 𝑂3°− + 𝐻+  → 𝑂𝐻° + 𝑂2   (1-13)  Figure 1.6  Mechanism of the direct and indirect ozonation (Gottschalk et al., 2010)  The OH° radical can react with ozone and decay to a hydroperoxyl radical (HO2°) and the chain reaction can start again. 𝑂𝐻° + 𝑂3  → 𝐻𝑂2° + 𝑂2   (1-14) Initiation Direct O3 O3 OH- M O2 Moxid M O2°-                                         HO2° O3°-                               HO3°    Chain Reaction OH° O2 Termination S   ? HO4° O3 R° ROO° O2 Moxid Indirect 28 In the chain reactions, the substances that can covert OH° into superoxide radicals O2°¯/HO2° are called promoters. Some organic molecules such as aryl-R, primary and secondary alcohols can act as promoters. Some substances such as carbonate and bicarbonate ions can react with OH°, and do not produce superoxide radicals (O2°¯/HO2°), thus terminating the chain reaction. These substances are called inhibitors or scavengers. 𝑂𝐻° + 𝐶𝑂32− → 𝑂𝐻− + 𝐶𝑂3°−  (1-15) 𝑂𝐻° + 𝐻𝐶𝑂3− → 𝑂𝐻− + 𝐻𝐶𝑂3°   (1-16) Table 1.2 shows some substances that act as initiator, promoter and terminator in the chain reaction. The direct reaction for ozone is selective and slower than the indirect radical chain reaction. Ozone tends to react faster with the unsaturated bonds and certain types of aromatic compounds. Ozone also reacts faster with the ionized or dissociated forms of organic compounds than with the neutral and non-dissociated compounds. Ozone can react with inorganic compounds at a faster rate than with organic compounds. Table 1.3 shows a comparison between direct and indirect reactions.  Table 1.2  Typical initiators, promoters and scavengers (Staehelin and Hoigne, 1983; Xiong and Graham, 1992) Initiator Promoter Scavenger OH¯ H2O2/HO2¯ Fe+2 Humic acid Aryl-R PO43-  Primary and secondary alcohols  HCO3¯/CO32- PO43-  Humic acid Alkyl-R Tert-butyl alcohol(TBA)  29 If the radical reactions are inhibited (no initiators or too many scavengers), direct reaction is important. Normally the pathways are also affected by pH. The direct reaction dominates under acidic conditions (pH < 4) and the radical reaction dominates if pH > 10. Table 1.3 Comparison between ozone direct reaction and indirect reaction  (Gottschalk et al., 2010) Reaction pathway Direct reaction Indirect reaction Oxidants O3  HO°  Selective? Selective --unsaturated bond more than saturated --aromatic more than  aliphatic  --dissociated more than undissociated --e- supplying substitutes more than e- detracting substitutes Non-selective Tend to reacts with the target molecule at the position with the highest electron density  Reaction rate Varies  KR = 10-3 ~ 109 /M/S, but generally slow  KR =107 ~109 /M/S, very fast  pH conditions dominant at low pH High pH Half life Long  Very short   When ozone is applied for water and wastewater treatment, direct and indirect reactions can occur at the same time and both pathways should be considered. In drinking water treatment, the concentration of O3, OH°, and intermediates such as O2°-, O3°- and organic radicals are assumed to be at steady state and the indirect reaction pathway (radicals oxidation) is dominant compared with the direct reaction (Gottschalk et al., 2010). However, in wastewater treatment, the concentration of dissolved ozone in the bulk liquid is assumed to be zero and the direct reaction is very important and the indirect reaction is negligible (Gottschalk et al., 2010) due to the presence of scavengers and the short half life of the HO°.  For sludge ozonation, the interactions between ozone and activated sludge are complicated and poorly understood because of the three-phase systems of sludge and the complex matrix of 30 sludge flocs, living cells and organo-mineral materials (Dziurla et al., 2005). Hydroxyl radicals generated during ozonation and other sludge disintegration technologies are assumed to be the critical factor for sludge solubilization and cell inactivation (Dziurla et al., 2005; Yan et al., 2009); however, the existence and effect of hydroxyl radicals has not been thoroughly studied due to the existence and generation of many scavengers in sludge. The study by Xu et al. (2010) reported that the ozone molecule reaction played a major role in sludge disintegration in a combination treatment of ultrasound and ozone, which was not consistent with other researcher’s results (Song et al., 2007).   1.4.2 Ozone Mass Transfer and Reactor Configuration The ozone mass transfer mechanism in gas/liquid systems is generally explained by the “fast kinetic regime” theory proposed by Danckwerts (1970). The ozone in gas/liquid systems has no mass transfer limitation in the gas phase due to its low solubility in water. The rate limitation occurs in the liquid phase or liquid film, where the ozone can react fast with the various dissolved substances and be consumed. This causes the apparent rate of ozone mass transfer to exceed the maximum rate of physical gas-liquid mass transfer. The mass transfer rate enhancement is characterized by the enhancement factor, E, which is defined as the ratio between the actual flux of ozone and the maximum flux due to physical absorption (Paul and Debellefontaine, 2007). 𝐸 = 𝑎𝑐𝑡𝑢𝑎𝑙 𝑓𝑙𝑢𝑥 𝑜𝑓 𝑜𝑧𝑜𝑛𝑒𝑚𝑎𝑥𝑖𝑚𝑢𝑚 𝑓𝑙𝑢𝑥 𝑑𝑢𝑒 𝑡𝑜 𝑝ℎ𝑦𝑠𝑖𝑐𝑎𝑙 𝑎𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑜𝑛 = 𝑟𝑂𝐾𝐿𝑎×(𝐶𝑂∗−𝐶𝑙𝑏) = 𝑟𝑂𝐾𝐿𝑎×𝐶𝑂∗  (1-17) Where,  31 𝑟𝑂 is the actual flux of ozone, quantified as the gas flow rate of ozone multiplied by the ozone concentration difference between the inlet and the outlet, mg/L/s;  𝐾𝐿𝑎 is the overall mass transfer coefficient, s-1;  𝐶𝑙𝑏 is the concentration of ozone in the builk liquid, mg/L, and, 𝐶𝑂∗  is the equilibrium concentration of ozone in liquid phase in equilibrium with bulk gas partial pressure , which is determined by Henry’s law,  mg/L.Figure 1.7 shows the profile of dissolved ozone concentration in the liquid film. Without enhancement by chemical reactions, the thickness of the liquid film is δ, and the ozone concentration decreases from the equilibrium concentration 𝐶𝑂∗  to 0. When dissolved organic or inorganic compounds are present in the liquid and react with the ozone which improves mass transfer, the effective thickness of the liquid film decreases as δE.       Figure 1.7  Schematic of profiles dissolved ozone in the liquid film (Foladori et al., 2010; Paul and Debellefontaine, 2007) Normally, the thickness of the liquid film, δ, is between 15 and 20 μm and with the enhancement by the fast reaction, the effective thickness of the film would be never greater than 10 μm; the liquid film is so thin that only smaller particles could be oxidized by ozone. (Paul and Gas Phase Liquid film Bulk liquid O3 concentration in gas phase 𝐶𝑂3∗  0 𝛿𝐸 𝛿 O3 concentration in bulk liquid OH°, HR°, HRO2° 32 Debellefontaine 2007).The flocs are partially attacked by ozone due to their large size and cells inside the flocs can remain viable due to the physical protection offered by the aggregate structures (Chu et al., 2009). It was also assumed that the hydroxyl radicals and dissolved ozone initially react very fast with most of dissolved substances, and are likely to be completely consumed before they encounter a dispersed target particle (Paul and Debellefontaine, 2007).  However, the “fast kinetic regime” film theory has flaws because it assumes that (1) mass transfer occurs through the film at steady state and (2) flux is low and the ozone concentration is low. It may not fully explain the mass transfer phenomenon in the gas/water/solid systems of activated sludge. According to the “fast kinetic regime” theory, as more soluble substances are released during ozonation at higher ozone dosage and the enhancement factor, E, increases and the thickness of the liquid film, δE, becomes thin, the rate of sludge solubilization (indicated by soluble COD increase) should increase. However, many experiments (Ahn et al., 2002; Nishijima et al., 2003; Sui et al., 2011) have shown that the rate of sludge solubilization becomes flat at high ozone dosage or long contact time. Another limitation associated with “fast kinetic regime” theory is that it can hardly explain the different degree of sludge solubilization under various configurations and operating conditions by applying the same dosage of ozone in the same sludge. In addition, the ozone dosage required to destroy cells is less than the amount of ozone needed to disrupt the flocs and reduce the particle size distribution (Foladori et al., 2010; Zhang et al., 2009), which indicates two possible scenarios: (1) the ozone direct reaction may occur beyond the liquid film at a certain dosage, which can inactivate the cells but not disintegrate the structure of flocs. (2) The ozone does not exist in bulk water but the radicals generated by ozone indirect reaction may migrate to the surface of sludge flocs and inactivated the viable cells. 33 1.4.3 Sludge Ozonation Reactor Configuration A high efficiency for sludge disintegration by ozone is required since the major constraint for sludge ozonation is the associated energy consumption and the operating cost. The performance for sludge ozonation is largely dependent on the reactor configuration and the operating parameter values, which are based on the ozone chemical reaction mechanism and mass transfer in the sludge. Mass transfer is the crucial factor to determine the configuration of an ozonation reactor  There are several kinds of reactors that have been used for sludge ozonation, such as bubble diffusers, impellers, static mixers and Venturi injectors. The most commonly used reactor for sludge ozonation is the ozone bubble diffuser contactor which is illustrated as Figure 1.8. In the ozone contactor, a rotating mixer is used to improve sludge mixing and contact with the ozone bubbles. Not all of the ozone will be utilized and the residual ozone in off-gas requires treatment in an ozone destructor. The ozone diffuser can be fabricated using oxidant-resistant materials such as ceramics, stainless steel, polyvinylidene fluoride (PVDF) or PTFE (polytetrafluoroethylene). Fine bubble- or micro-bubble diffusers can be used to produce large surface-area-to-volume ratio micro-bubbles and to improve ozone mass transfer (Chu et al., 2007). The depth of the ozone contactor column (from 61 mm to 183 mm) was observed (Egemen et al., 2001) not to significantly affect the solubilization efficiency. However, even when micro-bubbles are used, the gas transfer rate and efficiency in a sludge ozone contactor is still low. In addition, the sludge ozonation contactor configuration has serious foaming problems which become worse if the hydraulic retention time is longer.  Another widely used sludge ozonation reactor is the Venturi injector (Paul and Debellefontaine, 2007). A Venturi injector works by forcing liquid through a constricted section or choke of a 34 pipe, which creates a pressure reduction inside the injector body and initiates ozone gas suction through the suction port. The gas flow rate is an important factor affecting ozone mass transfer. It has been observed that ozonation with higher gas flow rate and lower ozone gas concentrations could achieve a greated extent of  sludge solubilization than a control treatment with the same ozone dosage (Manterola et al., 2008).   Figure 1.8  Sludge ozone treatment contactor (Park et al., 2002) Other sludge ozonation reactors have also been proposed. According to Henry’s law, increasing the partial pressure of the solute above a solution can enhance mass transfer from the gas phase to the liquid phase. Ozonation in pressure cycle reactors was reported (Cheng et al., 2012) to result in a significant increase in sludge disintegration. A turbulent jet flow ozone contactor (TJC) was tested by Hwang et al. (2010) indicating that  hydrodynamic cavitation can be generated and  used for sludge ozonation to maximize mass transfer efficiency of the ozone. A hybrid sludge ozonation approach, which involves a combination of sludge ozonation and another mechanical or chemical treatment, would be a feasible way to improve mass transfer and disintegration efficiency. In his thesis, Chanda (2012) reported  that the combination of ozonation and cavitation exhibited a higher degree of sludge disintegration than the individual methods applied alone. In another study (Yuxin et al., 2014), it was found that the efficiency of 35 sludge disintegration and the release of soluble COD, total nitrogen and total P by ozone/hydrogen peroxide (O3/H2O2) treatment was greater than that with O3 alone.  1.4.4 Ozone Dosage To quantify the amount of ozone applied to sludge during an ozonation process, ozone dosage is generally used as a key parameter. Ozone dosage is defined as the mass of ozone applied in the reactor divided by the mass of TSS in the sludge (Foladori et al., 2010), 𝐷𝑂3 = 𝑀𝑂3𝑉∙𝑥 = 𝑄𝑔𝑎𝑠∙(𝑂3,𝑖𝑛−𝑂3,𝑜𝑢𝑡)∙𝑡𝑄𝑠∙𝑥∙𝑡   (1-18) Where, 𝐷𝑂3 is the ozone dosage, g O3/g TSS; 𝑀𝑂3 is the mass of ozone applied, g; 𝑉 is the volume of reactor, L; 𝑥 is the TSS concentration, g/L; 𝑄𝑔𝑎𝑠 is the flow rate of gas, L/s; 𝑄𝑠 is the flow rate of sludge, L/s, 𝑂3,𝑖𝑛 is the concentration of ozone at the inlet of the reactor, g/L; and, 𝑂3,𝑜𝑢𝑡 is the concentration of ozone leaving the reactor, and t is the ozonation time, g/L. In some articles, the ozone dosage is also expressed normalized against the mass of VSS (g O3/g VSS) or the mass of COD (g O3/g COD).  The ozone dosage is the dominant factor determining the performance of a sludge ozonation system. Sludge solubilization depends strongly on the ozone dosage. The values of ozone dosage applied in many studies have ranged from 0.01 g O3/g TSS to 1 g O3/g TSS. The effect of ozonation on microorganisms and flocs as a function of ozone dosage is shown in Figure 1.9. 36  Figure 1.9  Effect of ozone dosage on sludge disintegration (Foladori et al., 2010)  An ozone dosage lower than 0.015 g O3/g VSS is insufficient to cause cell rupture (Albuquerque et al., 2008). It was reported that if the ozone dosage was below 0.02 g O3/g TSS, no alteration in bacterial DNA could be detected, which indicated that the DNA of sludge was not attacked by ozone (Yan et al., 2009). The destruction of bacteria occurs mainly at an ozone dosage greater than 0.08 g O3/g TSS (Yan et al., 2009). By measuring oxygen uptake rate (OUR), it has been observed that at an ozone dosage of around 0.02 g O3/g TSS, 80% of microbial respiration activity was lost (Chu et al., 2008). Another study (Saktaywin et al., 2005) reported that around 70% of sludge bacterial activity was lost at an ozone dosage of 0.03-0.04 g O3/g TSS. It was also reported that the total protease and catalase activities were gradually reduced at ozone dosages from 0 to 0.05 g O3/g TSS, which indicated that enzymes began to lose their activity after the addition of ozone (Yan et al., 2009). An increase in soluble extracellular polymeric substances (EPS) was observed at ozone dosages from 0.022 to 0.070 g O3/g TSS (Dytczak et al., 2007). Ozone may destroy part of the flocs, such that a fraction of the bound polymeric material is released and solubilized. Only at higher 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0.11 0.12 0.13 0.14 0.15 0.16 0.17 0.18 0.19 0.2Ozone dosage (gO3/gTSS) No alteration of DNA Bacteria begin to break downn Reduction of activity and respiration by 70~80% Destruction of bacteria Reduction of particle size Flocs broken into dispersed particles Decrease of enzymatic activity Increase of soluble EPS Inactivation of fecal coliforms 37 dosages of 0.088 g O3/g TSS does ozonation cause a decrease in  the amount of EPS (Foladori et al., 2010). Ozone has little effect on sludge floc structure. Some studies (Zhang et al., 2009) reported that the particle size distribution in sludge does not change significantly after 0.05 g O3/g TS dosage ozone treatment, which indicates that ozone does not disintegrate floc structure at lower dosages. Only at higher dosage (> 0.1 g O3/g TS), does the mean particle size gradually move to a smaller value (Park et al., 2003). At a dosage of 0.16 mg O3/g TS, the flocs were observed to have been broken into fine and dispersed particles (Chu et al., 2008).  At higher ozone dosages, the extent of mineralization rises and solubilized organics decrease (Yeom et al., 2002). At an ozone dosage of 0.1 g O3/g TSS, approximately 5% of total solids mass may be mineralised (Foladori et al., 2010). The mineralization of sludge reached 14% and 26% when the ozone dosage was increased to 0.18 and 0.27 g O3/g TSS respectively (Yan et al., 2009). Sludge solubilization depends strongly on the ozone dosage. TSS disintegration occurs due to solubilization and mineralization. TSS disintegration increased in an almost linear fashion at the lower dosage of up to 0.04 - 0.05 gO3/TSS and the degree of solubilization was in the range from 2 to 6 g TSSsolubilised/g O3 (Nagare et al., 2008; Sakai et al., 1997). For ozone dosages higher than 0.05 g O3/g TSS, the COD solubilization rate increases slowly because in this range the amount of COD solubilized is of a similar magnitude to that of the soluble COD oxidized by ozone. When the ozone dosage was 0.08 g O3/g TSS, a higher portion of ozone reacted with dissolved organic matter (Zhang et al., 2009). When the ozone dosage was above 0.14 g O3/g TSS, the ozonation efficiency decreased due to the release of radical scavengers such as lactic acid and sulfate ions from sludge (Yan et al., 2009). At  relatively high ozone dosages above 0.2 g O3/g 38 TSS, the efficiency of sludge disintegration and solubilization does not change significantly (from 45% to 65%) (Foladori et al., 2010).  The optimal ozone dosage represents a compromise between ozonation costs, excess sludge treatment and disposal costs and the maintenance of the wastewater treated effluent quality required. Higher ozone dosages produce less excess sludge; however, high dosages can lead to mineralization and can decrease the biodegradability of sludge. Moderate ozone dosage is required to convert solids into easily biodegradable COD to facilitate subsequent removal in a biological treatment process.   1.4.5 Summary Among the available sludge disintegration technologies, ozonation has been shown to be effective in achieving a high extent of sludge solubilization. The major drawback for ozonation is its high energy requirement. The performance (the degree of sludge solubilization and energy requirement) for sludge ozonation is dependent on the reactor configuration and the process operating parameters, which in turn, are based on the ozone chemical reaction mechanism and the mass transfer achieved in the sludge. However, the chemical reaction mechanism is poorly understood for sludge ozonation because the interactions between ozone and activated sludge are complicated by the three-phase nature of sludge and the complex matrix of sludge flocs which includes living cells and organo-mineral materials. It has not been identified which pathway (ozone direct oxidation or radicals indirect reaction) is dominant in sludge ozonation. Mass transfer is also crucial in sludge ozonation. The hybrid disintegration method, which is a combination of ozonation with mechanical treatment methods, could be a feasible approach to 39 improve mass transfer and thus, achieve a high degree of sludge solubilization with moderate energy consumption.  1.5 Sludge Ozonation Coupled to Biological Wastewater Treatment 1.5.1 Sludge ozonation in conventional secondary treatment Ozonation has been assessed as an option to minimize sludge production in conventional activated sludge treatment because it can not only solubilize particles but also mineralize sludge. A secondary wastewater treatment process combined with sludge ozonation was first proposed by Yasui and Shibata (1994). A full-scale combination of an activated sludge process with sludge ozonation, without excess sludge wasting, was reported by Yasui et al. (1996). About one-third of the ozonated sludge was mineralized and then returned to the influent as feed, and thus, no excess sludge was wasted. The conventional secondary wastewater treatment system combined with ozonation to reduce sludge production was then further studied by several researchers (Kamiya and Hirotsuji, 1998; Lee et al., 2005; Sakai et al., 1997; Yasui et al., 1996). In the application of ozonation to reduce sludge production in an industrial wastewater treatment plant, one study (Albuquerque, Domingos et al. 2008 showed that ozonation was able to reduce sludge production by 14 to 39% when 20% of the recycle sludge was ozonated with a recycle ratio of 0.67.  Although the operating cost for sludge ozonation is comparable to that of conventional sludge treatment and sludge disposal (Yasui et al., 1996), the dosage of ozone required  to minimize excess sludge production is quite high and the associated energy requirement is significant.  40 Previous research has indicated that the effluent quality parameters COD and SS were not significantly affected (Kamiya and Hirotsuji, 1998; Lee et al., 2005; Sakai et al., 1997; Yasui et al., 1996) by ozonation. On the contrary, although the temporary ozone treatment of sludge was observed to have negligible effects on the heterotrophic biomass (Gardoni, 2015), sludge ozonation was found to decrease the COD removal performance. The refractory total organic carbon (TOC) and SS in the effluent increased after sludge ozonation, which may be due to refractory TOC and fine particulates release with sludge ozonation and the short actual SRTs.  Inorganic substances in mixed liquor volatile suspended solids (MLSS) tend to accumulate with sludge ozonation. If the recirculated sludge is not properly treated, inert particulate COD and inorganic TSS present in the influent may accumulate in the reactor, resulting in a decrease in the VSS/TSS ratio and a reduction of the active biomass fraction. The VSS/TSS ratio has been observed to decrease (Sakai et al., 1997; Yasui et al., 1996) compared with a control process. The concentrations of some metals such as Fe/Al in the sludge increased while some (Ca/Mg) remained constant after ozonation for zero sludge production (Sakai et al., 1997; Yasui et al., 1996).  The settleability of ozonated mixed liquor was reported to improve as noted by several researchers (Nilsson et al., 2014; Romero et al., 2015; Sakai et al., 1997; Yasui et al., 1996; Yasui and Shibata, 1994). The sludge volume index (SVI) was found to decrease with sludge ozonation and good sludge settling characteristics were maintained. The filamentous bacteria were inactivated after ozonation and the networks of filamentous bacteria almost disappeared.  The concept of a zero sludge wastewater treatment system has been investigated by several authors (Lee et al., 2005; Sakai et al., 1997; Yasui et al., 1996). In a zero sludge system, since no excess sludge is wasted, the apparent SRT is infinitely long. However, the actual SRT is limited 41 because biosolids are partially solubilized and cells are inactivated during the sludge disintegration process. Theoretically, the amount of VSS solubilized during sludge disintegration should be equal to the amount of VSS that otherwise would be wasted from a traditional activated sludge treatment system. However, in practice, since current sludge disintegration technologies cannot solubilize 100% of the TSS, the recirculation rate for sludge ozone treatment should be higher than the sludge withdrawal rate in traditional activated sludge treatment. It has been reported (Yasui et al., 1996) that a recirculation rate of 3.3 times the sludge withdrawal rate was required to achieve zero sludge production. The required rate may vary depending on many factors such as influent characteristics, operating conditions, sludge characteristics and sludge disintegration system efficiency. Another problem for zero sludge wastewater treatment systems is that phosphorus cannot be removed if there is no excess sludge withdrawn. Some authors (Saktaywin et al., 2005) proposed the application of  sludge ozonation to release PO4-P and then recover it by crystallization; however, the soluble PO4-P achieved was limited with ozone dosage increase and polyphosphate was the main component of solubilized phosphorus with sludge ozonation and polyphosphate was difficult to  directly recover.  1.5.2 The Effect of Sludge Ozonation on Nitrification and Biological Phosphorus Removal  For the biological nutrient removal (BNR) process combined with sludge ozonation to minimize sludge generation, very few studies have been reported. Nonetheless, it might be expected that BNR performance may be reduced with sludge ozonation. Nitrification may be inhibited by ozonation treatment due to the direct influence of ozone on nitrifying bacteria and the prevailing process SRT may be too short to maintain nitrification. However, some researchers (Bohler and 42 Siegrist, 2004) have indicated that the reduction of the activity of the nitrifiers due to ozonation may be compensated by an increase in apparent SRT due to the lower excess sludge production. For denitrification, it was observed to be enhanced, rather than inhibited, by ozonation in a comparative study (Dytczak et al., 2007) in which  two nitrifying sequencing batch reactors (SBRs) were operated coupled to sludge ozonation. The study also indicated that ozonation of 20% of recycle activated sludge (RAS) had no negative impact on the final effluent quality (COD and TSS) and that nitrification was complete in both reactors even with the additional ammonia released by ozonation. The denitrification rate increased by 60% due to the extra supply of soluble COD with ozonation. However, some accumulation of inorganic solids in the ozonated reactor was observed. It seems that a low dosage ozone sludge treatment in an enhanced biological phosphorus removal (EBPR) process did not have an inhibitory effect on COD removal and nitrification and denitrification, but P removal was affected. In a 500 m3/d wastewater treatment plant with the A/A/O process, a low dose of ozone was introduced by a Venturi nozzle to treat recycled sludge. The authors reported that no sludge cell rupture was observed and the performance of the A/A/O process was improved (Meng et al., 2013). In a comparative bench scale study of an extended aeration activated sludge process and an activated sludge process coupled to an ozonation system (Demir and Filibeli, 2014), it was shown that the COD removal efficiency and the nitrification capacity were not significantly decreased, but that phosphorus removal efficiency was reduced in the coupled system. In a long-term full-scale sludge reduction study by Gardoni et al. (2011), a wastewater treatment plant coupled with a short contact time sludge ozonation unit achieved 17% excess sludge reduction and the decay rate of the heterotrophic increased from 0.62 d-1 to 1.3 d-1 and the heterotrophic growth yield value decreased from 0.68 g COD/g COD  to 0.58 g COD/g 43 COD. However, the N removal efficiency decreased from 58% to 46% due to a reduction of simultaneous nitrification-denitrification reactions associated with the higher dissolved oxygen concentration in the aerobic reactor after sludge ozonation. In a pilot study (Sui et al., 2014) on sludge reduction by ozonation and phosphorus recovery in an advanced sewage treatment process, the results showed that nitrogen removal by denitrification increased, nitrogen in the effluent did not change and the reduced nitrogen in excess sludge was transformed to nitrogen gas by denitrification. In another pilot-scale study (Qiang et al., 2015), the authors investigated the long-term performance of an anaerobic/anoxic/oxic (A/A/O) process coupled with sludge ozonation and phosphorus (P) recovery. The system achieved a maximum sludge reduction efficiency of 85%. They also concluded that sludge ozonation had little effect on COD and N removal, MLVSS/MLSS ratio, and sludge activity in the bioreactor.  Few research articles have reported a focus on phosphorus removal after sludge ozonation, but the reported effects of sludge ozonation on the activity of PAOs (phosphorus-accumulating organisms) are contradictory according to the literature. One full-scale study (Gardoni et al., 2011) concluded that P removal efficiency decreased from 70% to 28% due to reduced biosynthesis of PAOs. In a pilot-scale study, the effluent TP concentration increased and P removal was inhibited (Qiang et al., 2015). However, in a study (Nilsson et al., 2014) using ozone to reduce filamentous bulking in a full scale WWTP, the researchers found that the application of ozone did not affect biological P removal efficiency. In another study (Lee et al., 2011), ozone lysate of excess sludge was used as a carbon source for phosphorus removal from low strength wastewater in a modified intermittently decanted extended aeration (IDEA) process, and the removals of nitrogen and phosphorus were found to be simultaneously enhanced. 44 According to the study, although the ozone lysate was less effective than acetate for phosphorus release, it was still a satisfactory carbon source for complete removal of the phosphorus.  In summary, very few articles have focused on sludge ozonation combined with advanced wastewater treatment and for the few studies reported there is no general consensus reached on the effect of sludge ozonation on nutrient removal, especially phosphorus removal, which needs further study.    1.6 Knowledge Gap Since both a very long SRT operation and sludge ozonation can achieve sludge reduction, if the two approaches are combined, a synergistic effect may be achieved and even greater sludge reduction might be obtained. The combination of sludge ozonation and very long SRT operation in an activated sludge process has seldom been reported. Although the integration of ozone systems in a biological treatment process has been reported and demonstrated to achieve a high degree of sludge solubilization and sludge reduction (Arakawa et al., 2011; Deleris et al., 2002; Inchauste-Daza et al., 2011; Romero et al., 2015), the high energy requirement and the associated high operational cost is the major drawback. Nonetheless, ozone treatment and partial oxidation by ozone at a low dosage coupled to extensive biological oxidation (Ried et al., 2007) would be desirable. It is well known that sludge production is low in an activated sludge process operated at a long SRT because endogenous metabolism reduces biomass production. It is possible that a combination of sludge ozonation and long SRT operation may reduce sludge yield even with a low dose of ozone that might be economically feasible. The improved sludge settling properties with ozone treatment reported by a few researchers (Chiellini et al., 2014; Lyko et al., 45 2012) also supports long SRT operation in an activated sludge process coupled with sludge ozone treatment. With more stringent effluent standards and higher ambient water quality reqirements, many traditional secondary wastewater treatment plants have upgraded to the enhanced biological phosphorus removal (EBPR) process. Base on the literature reviewed, the effect on microbial activity during sludge reduction by ozonation in an EBPR process has not been thoroughly studied. Although a few articles have reported that nitrification would not be affected and the lysate generated by sludge ozone treatment may be used as carbon source to enhance denitrification (Inchauste-Daza et al., 2011; Park et al., 2011; Yang et al., 2011), the effect on the activity of the phosphorus-accumulating organisms (PAOs) and kinetics of biological phosphorus removal are still controversial and no general consensus has been reached.  To reduce ozone dosage and  operating costs , it is necessary to optimize the sludge ozone treatment process. There are few studies that are focused on mixed liquor ozonation combined with biological wastewater treatment. Many researchers (Gardoni et al., 2015; Inchauste-Daza et al., 2011; Park et al., 2011; Yasui et al., 1996) used ozone to disintegrate the excess sludge from a secondary clarifier and then recycled the lysate as a carbon source to the biological wastewater treatment process. With high suspended solids concentration, the return sludge from the secondary clarifier may consume high dosages of ozone, which could reduce the efficiency of ozonation. It was reported (Gardoni, 2015) that high TSS concentrations of sludge may reduce COD solubilization efficiency by a Venturi plug-flow rector and lower TSS or MLSS may be preferred for sludge ozone treatment to improve the efficiency. It is possible that mixed liquor ozonation may be more efficient and may require a lower ozone dose than recycle sludge ozonation. The chemical reaction mechanism for sludge ozonation is poorly understood and the 46 interactions between ozone and activated sludge are complicated due to the three-phase system of mixed liquor and the complex matrix composed of sludge flocs, living cells and organo-mineral materials. Mass transfer is crucial in sludge ozonation. The hybrid disintegration method, which is combined ozonation with mechanical or other chemical treatment methods, could be a feasible approach to improve mass transfer and thus, achieve a high degree of sludge solubilization with low energy consumption. To facilitate wastewater treatment process modelling and design for sludge reduction by mixed liquor ozonation, the kinetic parameters need to be measured. Although a few authors (Isazadeh et al., 2005; Wang et al., 2008; Yoon, 2003) proposed basic mathematical models to describe the mass balance of wastewater treatment combined with sludge ozonation, the heterotroph kinetic parameters such as decay rate and maximum growth rate have not been measured. These parameters are crucial to construct a dynamic model to simulate the process and to estimate the suspended solids production when sludge ozonation is combined with a biological wastewater treatment process. The zero sludge EBPR system would be the ultimate goal of sludge reduction. The concept of a zero sludge wastewater treatment has not been achieved for a enhanced biological phosphorus removal(EBPR) process, although previous researchers have concluded  that zero sludge production could be achieved in conventional activated sludge wastewater treatment systems combined with sludge ozonation and mineralization (Sakai et al., 1997; Yasui et al., 1996). It may be difficult to achieve zero sludge for BNR processes, due to the complexity of the system, the sensitivity of the process microorganisms, and the consequences of phosphorus accumulation in the reactor.  47 1.7 Objective of the Study This study focused on the following research questions. (1) Can significant excess sludge reduction in an EBPR system be achieved by combining long SRT operation with mixed liquor ozone treatment?  A laboratory-scale EBPR reactor coupled with a mixed liquor ozone treatment unit was constructed and tested with a municipal wastewater feed. Two different SRTs (25 days and 50 days) were assessed by comparative study. The ozonation was applied to mixed liquor rather than recycled sludge to avoid foaming problems. (2) Would the effluent quality be affected? What is the impact on nitrification and denitrification? What is the impact on phosphorus removal?  The comparative study and batch tests were conducted to investigate the effect of sludge ozonation on phosphorus removal and activity of PAOs in an EBPR process. (3) What’s the effect on microorganism activity and microbial community structure if mixed liquor ozone treatment is combined with the EBPR system? Can the microbial community adapt to the mixed liquor ozonation in the EBPR system? The mixed liquor was sampled, observed and tested regularly to exam the microorganism activity and microbial community. Molecular tools were used to study the changes after sludge ozonation. (4) What are heterotrophic kinetic parameters such as the decay rate, maximum growth rate if mixed liquor ozonation is combined with biological wastewater treatment?  48 The heterotrophic kinetic parameters were measured by batch tests. The results of the measurements can be used to build a model and estimate the solids production during sludge ozonation in a wastewater treatment process. (5) Would a zero sludge EBPR system be possible if phosphorus is extracted from the system? Although a zero sludge wastewater treatment system has been achieved for conventional activated sludge processes, no zero sludge EBPR processes have been reported. In this study, by an experimental approach, phosphorus removal and extraction from the EBPR process combined with mixed liquor ozonation was studied.    49 Chapter 2: Materials and Methods  2.1 The Experimental Setup 2.1.1 Two SBR-MBR Systems  Two identical 20-liter sequencing batch reactors (SBR) were built, each followed by a membrane tank for final solids-liquid separation. One served as a control (Figure 2.1), and the other was combined with a mixed liquor ozone treatment unit (Figure 2.2). The valves, the pumps and the mixer for the SBR-MBR systems and the ozone reactor were operated automatically by a controller. The SBR-MBR systems mainly included four units: (a) The SBR tank to remove organic contaminants and nutrients; (b) membrane tank for solids-liquid separation of the effluent from the SBR; (c) A sludge ozone treatment reactor; and (d) the controller.  50  Figure 2.1   The schematic diagram of the SBR-MBR system for the control 51  Figure 2.2   The schematic diagram of the SBR-MBR system combined with a mixed liquor ozone treatment reactor  52 2.1.2 The SBR Configuration The sequencing batch reactor (SBR) main tank was made of acrylic (poly(methyl 2-methylpropenoate)) tube and acrylic flat sheet (Figure 2.3 and Figure 2.4). The height of the tank was 45.7 cm (18 inch) with 30.5 cm (12 inch) external and 29.2 cm (11.5 inch) internal diameters. There were six ½ inch NPT ports opened in the tank: the outlet for mixed liquor ozone treatment, the inlet for treated sludge at the bottom, the port for the wastewater influent, the outlet for the effluent, the inlet for the VFA feed, and the outlet for overflow at the top, respectively. The liquid level was set to 30.5~38.1 cm (12~15 inches) depending on the operational requirement. The total liquid volume of the tank was about 20~25 liters. The volumetric filling ratio of the SBR could be set from 25% to 40%.  Figure 2.3 The diagram of the sequencing batch reactor 53  Figure 2.4 The photo of the SBR A 20.3 cm (8 inch) diameter aeration disc was installed at the bottom of each SBR tank. On the top of the tank, there was a 5.1~12.7 cm (2-5 inch) blade mixing impeller with a variable-frequency drive (VFD) controller. The flow rates of the influent and the VFA feed were controlled by three peristaltic pumps. The effluent was discharged by opening an air actuated ball valve, which was controlled by a solenoid valve.  2.1.3 The Membrane Tank The membrane tank was installed after the SBR tank for further purification of the effluent of the SBR. The membrane tank tank was made of an acrylic cylindrical column with 1.5 m height and 14 cm diameter. Figure 2.5 shows the configuration of the membrane tank.  54  Figure 2.5 The schematic diagram of the membrane tank There were three ports in the tank: an inlet for the effluent from the SBR in the middle, a sludge outlet at the bottom, and an overflow port at the top. An L-shaped pipe was connected to the main cylindrical column for water level control. A water level sensor in the L-shape pipe was linked to the controller. 55 A hollow fiber membrane module ZeeWeed®-10 was used for solids-liquid separation. It stood at the bottom of the tank vertically and was held in place with a frame clamped to the aeration tube. The module dimensions are shown in Figure 2.6. A ZW10 module comes with an extended aeration tube and two holes on the top header: one for permeation and one for pressure measurement. Permeation was done only from the top header. The aeration tube supplied air to the bottom header where air diffusers were located. The membrane module specifications were as follows, • nominal membrane surface area 0.93 m2 (10 ft2), • membrane type MF200, • permeation rate to the desired value: 0-100 L/m2∙h (0-60 gal/ft2/day), • membrane surface chemistry neutral and hydrophilic, • nominal membrane pore size 0.1 μm, • typical operating TMP 10-50 kPa (1.0-7.0 psi) @ 40°C (104°F), and • maximum aeration flow per module 3.6 m3/h (2 SCFM).  Figure 2.6 Diagram of ZeeWeed®-10 membrane module (Zenon Environmental Inc.)  56 2.1.4 The Mixed Liquor Ozonation Reactor The mixed liquor ozone treatment system included: (1) an oxygen cylinder and ozone generator; (2) a sludge ozonation reactor; and (3) an automatic control system. The system is shown in Figure 2.7 and the key components are shown in Figure 2.8.  The ozone generator was a model VMUS 08 made by Azco Industries Limited of Surrey, BC, Canada. Pure oxygen was used for ozone generation. The oxygen and its cylinder were supplied by Praxair Technology Inc. The sludge ozone reactor was composed of a positive displacement pump, a Venturi injector, a coil mixer, a bypass valve, and a pressure transducer. The positive displacement pump was a Moyno 331 with a ½ HP motor. The pump curve and operating point are illustrated in Figure 2.9. The pump was connected to the SBR tank by a ½ inch pipe. The flow rate was set to around 3.0 L/min and the pressure was 0.31-0.34 Mpa (45-50 PSI). The ozone injector was an in-line Venturi injector which was a Mazzei® Model 283 with compression fittings. A parallel bypass pipe with a valve was connected with the injector to prevent system damage if the pipe became blocked. The coil mixer that followed the ozone injector provided further retention time for the mixing and reaction of ozone and sludge. The tubing coil was made of 91.4 cm (3 feet) PFA (perfluoroalkoxy alkanes) tubing with ½ inch internal diameter. The outer diameter of the coil mixer was 10.2 cm (4 inch). The outlet of the coil mixer was connected to the bottom of the SBR tank. During sludge ozonation treatment, mixed liquor was pumped from the SBR, mixed and reacted with the injected ozone, and then sent back to the SBR tank. 57  Figure 2.7 Diagram of the sludge ozonation treatment system  Figure 2.8 Photos of the main components of the sludge ozonation treatment system      (a) the mixing tubing coil;     (b) the Venturi ozone injector and the bypass valve;     (c) the Moyno 500 series pump 58  Figure 2.9 The curve of the positive displacement pump curve and the operating point  The power of the pump, the valve for the oxygen gas and the ozone generator were controlled by the switch controller, which received signals from the programmable controller of the SBR-MBR system and the pressure transducer. The controller of the SBR-MBR sent requests to start and stop sludge ozonation. The pressure transducer was installed along the pipe between the pump and the ozone injector to monitor the pressure during operation for purposes of safety. If the pressure in the pipe reached a specified threshold value (say, 60 PSI), the sludge ozonation system was shut down by the controller, based upon the signal sent by the pressure transducer.  2.1.5 The Controller All processes of the SBR-MBR system were automatically controlled by a Programmable Logic Controller (PLC), the Siemens® logic module LOGO! The control logic is shown in Figure 2-10. The devices under control were the feed pump, the propeller mixer in the SBR tank, the pump for volatile fatty acids (VFA) addition, the valve for aeration, the solenoid valve for actuation of 59 the SBR discharge ball valve, the membrane permeate pump, the valve for air sparging on the membrane, and the sludge ozonation switch. By turning on and off these devices, the SBR-MBR system operated with the following steps (stages) in each cycle: feed (6-20 min), anoxic reaction (30-60 min), anaerobic reaction (60-90 min), the 1st aerobic (90-150 min) reaction, mixed liquor ozone treatment (1-2 min), anoxic reaction (10-50 min), the 2nd aerobic reaction (50-75 min), settling (25-50 min), SBR effluent discharge (about 25 min), MBR permeate (to lower water level), and idle (depending on the total cycle time).  Figure 2.10 The control logic of the SBR-MBR system with mixed liquor ozonation  60 2.2 The SBR-MBR Operational Conditions 2.2.1 The Wastewater The experimental equipment was located at the UBC wastewater treatment pilot plant and the feed to the SBR-MBR system was wastewater diverted from the UBC sewer, which carries sewage primarily from two residential areas at the east side of the UBC campus with a combined population of about 5000 to 6000 residents. A sump (Ф1.2 m×1.2 m depth) was connected to the sewer, from which wastewater was pumped to three storage tanks with 9.46 m3 volume connected in series for the purpose of influent flow equalization. Sodium bicarbonate was added once per day to the raw sewage storage tanks to maintain a stable pH 7.0. The wastewater in the storage tanks was then pumped to a primary clarifier with a surface overflow rate of 34.6 m/d and an HRT of 0.6 h. The effluent from the primary clarifier was pumped as the feed to the two SBR-MBR systems. The influent characteristics were monitored daily and a summary of the data is shown in Table 2.1.   Figure 2.11   UBC Pilot Plant and Storage Tanks for Wastewater  61 Table 2.1  Influent characteristics Contaminants Unit Concentration Low  Medium  High Temperature ℃ 12 18 24 TSS mg/L 32 82 381 VSS mg/L 19 72 338 COD mg/L 144 290 574 NH3-N mg/L 12 39 56 PO4-P mg/L 0.3 2.8 7.2  2.2.2 Experimental Phases The operating conditions of both SBR-MBRs were set to the same values in order to compare the performance and sludge generation with the two processes. Sludge was wasted daily for SRT control and waste volumes were adjusted weekly according to the most recent weekly aggregated suspended solids in the entire system to achieve the target SRT. The mixed liquor temperatures in the reactors were not controlled and the process temperatures varied between 12 and 24ºC during the period of the study, which lasted more than 400 days for three experimental phases completed.  Phase I. SRT = 25 d. The sludge retention time (SRT) was 25 days and the hydraulic retention time (HRT) was 24 hours. The two SBR-MBRs ran 4 cycles per day and each cycle was 6 hours. During the anaerobic reaction stage, about 150-200 mL sodium acetate solution (14 g/L) was added to the reactors to promote phosphorus-accumulating organisms (PAOs) growth.  Phase II. SRT = 50 d. The sludge retention time (SRT) was 50 days and the hydraulic retention time (HRT) was 24 hours. The two SBR-MBRs ran 4 cycles per day and each cycle was 6 hours. During the anaerobic reaction stage, about 150-200 mL sodium acetate solution (14 g/L) was added to the reactors to promote phosphorus-accumulating organisms (PAOs) growth. 62 Phase III. No Sludge was wasted.  2.2.3 The Operating Conditions in Phase I and II The comparative experiment was run from June 26th, 2015 to April 28th, 2016 and the total operational time was 311 days. There were two experimental phases: (I) nominal SRT = 25 days and (II) nominal SRT = 50 days. The system ran for 102 days at the first condition (nominal SRT = 25 d) and for 209 days at the second condition (nominal SRT = 50 d). The two experimental runs were completed independently.  After the first run (Phase I), mixed liquor from both reactors was mixed and divided equally to each reactor prior to the second run. This was followed by a transitional period (about 30 days) to allow the mixed liquor to adapt and accumulate in both reactors. There were two runs (i and ii) during experimental Phase II (nominal SRT = 50 d) using different substrates in the feed and operating with different F/M ratios. During Run i, 2 mL/d “cocktail” was added to the control reactor and 2 mL “vehicle” was added to the ozone-treated reactor every day. Both “cocktail” and “vehicle” were composed of 800 mL of 95% ethanol and 200 mL of 100% methanol for a 1 L solution. The purpose of adding cocktail and the vehicle was to generate treated effluent for a related, but independent, study on emerging contaminants. VFA for P removal was also added.  During Run ii, no vehicle or cocktail were added, and wastewater from the UBC campus and VFA for P removal were the only substrates for both reactors, which were the same as the substrates applied during Phase I (nominal SRT = 25 d). The operational conditions of the two SBRs are summarized in Table 2.2.   63 Table 2.2  Operational conditions for the control and the ozone-treated SBRs  The control The ozone treated SBR Stages 1. Fill: 5-6 minutes 2. Anoxic reaction:  about 50 minutes 3. Anaerobic stage : about 50 minutes  4. The 1st Aeration: 70-90 minutes 5. Idling: 1 minute 6. Anoxic Mixing: about 10 minutes 7. The 2nd Aeration: 60-90 minutes  8. Settle: 30-40 minutes 9. Decant: about 15 minutes  10. Membrane filtration: 20-25 minutes 11. Idling Total: 360 minutes 1. Fill: 5-6 minutes 2. Anoxic reaction:  about 50 minutes 3. Anaerobic stage : about 50 minutes  4. The 1st Aeration: 70-90 minutes 5. Ozone treatment (1 minute). 6. Anoxic Mixing: about 10 minutes 7. The 2nd Aeration: 60-90 minutes  8. Settle: 30-40 minutes 9. Decant: about 15 minutes  10. Membrane filtration: 20-25 minutes 11. Idling Total: 360 minutes HRT and SRT HRT = 24 hours Nominal SRT = 25 d (102 days)  Nominal SRT = 50 d (209 days) HRT = 24 hours  Nominal SRT = 25 d (102 days) Nominal SRT = 50 d (209 days) F/M ~ 0.16 g COD/ g VSS∙d (SRT=25 d) ~ 0.12 g COD/ g VSS∙d (SRT = 50 d, Run i) ~ 0.09 g COD/ g VSS∙d (SRT = 50d, Run ii) ~ 0.22 g COD/ g VSS∙d (SRT=25 d) ~ 0.13 g COD/ g VSS∙d (SRT=50 d, Run i) ~ 0.10 g COD/ g VSS∙d (SRT=50 d, Run ii)  To support an EBPR process, the SBR operation included anoxic, anaerobic and aerobic stages. Mixed liquor ozone treatment was not conducted during the anoxic phase or the anaerobic phase because (1) the soluble COD in the mixed liquor could consume large quantities of ozone and reduce the efficiency of sludge ozonation; and (2) the dissolved oxygen (DO) generation and oxidation reduction potential (ORP) increase might disturb the anaerobic and/or anoxic conditions. As mentioned in Chapter 1,  ozone transfers and reacts quickly with the dissolved substances in the mixed liquor, which is generally explained by the “fast kinetic regime” theory proposed by Danckwerts (1970); therefore, ozone treatment after most of the biodegradable 64 COD had been consumed by the microorganisms was preferred. Since batch testing indicated that more than 95% of the biodegradable COD was consumed after about 60 minutes of aerobic reaction, the mixed liquor ozone treatment was started after 70-90 minutes of aerobic reaction, followed by mixing and a further 60-90 minutes of aerobic reaction (the 2nd aeration) to degrade the lysate generated by the ozone sludge treatment. The hydraulic retention time (HRT) was 24 hours for both reactors. The nominal SRT = 25 d and SRT = 50 d were maintained by wasting mixed liquor from the SBRs during the aerobic stage. As noted above, there was a transition period between the two experimental phases at different SRTs which lasted about 30 days without sludge wasting, to adapt and accumulate sludge in the reactor. The food/mass (F/M) ratios differed with the experimental phases and runs: 0.16 ~ 0.22 g COD/g VSS∙/d for Phase I with SRT = 25 d,0.12 ~ 0.13 g COD/g VSS∙/d during Run i for the Phase II with the SRT =50 d and 0.09~0.10 g COD/g VSS∙d during Run ii for Phase II with the SRT = 50 d. The generally higher F/M for the ozone-treated SBR than for the control was due to the lower biomass concentration in the ozone-treated reactor.  2.2.4 SRT Control Solids retention ime (SRT) was maintained by wasting sludge from the SBRs daily. It was assumed that all suspended solids from the effluent of each SBR were retained in the MBR tanks and therefore, no suspended solids were present in the permeates of the membrane filtration units. The suspended solids accumulated in the MBR tank and from there, they were discharged and cleaned every 1-2 weeks. The volume of sludge to be wasted was determined by the sludge mass balance in equation 2-1. 65 V𝑆𝐵𝑅𝑆𝑅𝑇𝑋� = 𝑊𝑋� + 𝑉𝑀𝐵𝑅𝑋𝑀𝐵𝑅∆𝑇                      (2-1) Then,  𝑊 = 𝑉𝑆𝐵𝑅𝑆𝑅𝑇+ 𝑉𝑀𝐵𝑅𝑋𝑀𝐵𝑅∆𝑇𝑋�                               (2-2) Where V𝑆𝐵𝑅 = The volume of the SBR tank, L, 𝑆𝑅𝑇 = Nominal solids retention time, d,  𝑋� = The average concentration of mixed liquor suspended solids (MLSS), mg/L, 𝑊 = The volume of sludge to be wasted from the SBR daily, L, 𝑉𝑀𝐵𝑅 = The volume of the MBR tank, L, 𝑋𝑀𝐵𝑅 = The suspended solids concentration in the MBR tank, mg/L, and ∆𝑇 = The time period of the membrane tank cleaning (every 1-2 weeks), d. Using Equation 2-2, the volume of sludge to be wasted daily from the SBR tank was calculated after estimating the average concentration of SBR MLSS and the suspended solids concentration in the MBR tank.   2.3 Sludge Ozonation Batch Test  To investigate the sludge disintegration efficiency, three types of sludge were tested: (1) mixed liquor from the aerobic tank of the UBC MEBPR (membrane enhanced biological phosphorus 66 removal) pilot plant operating with a 40 day SRT, (2) the mixed liquor from the aerobic phase of the control SBR (20 L lab-scale) at SRT = 50 d, and (3) the mixed liquor from aerobic phase of the SBR (20 L lab-scale) combined with a sludge ozone treatment unit at a nominal SRT = 50 d.  Mixed liquor from the UBC MEBPR pilot plant contained a high suspended solids concentration (TSS = 11,580 mg/L). Therefore, it was diluted by 75% and 50% with its membrane permeate (effluent) from the same process to generate two different suspended solids concentrations for sludge ozonation testing.  Table 6.1 shows the types, compositions and characteristics of the sludges utilized for ozone treatment.  The sludge samples from the aerobic phase of the control SBR were prepared and completely mixed as: (1) 2 L mixed liquor + 500 mL distilled water, (2) 2 L mixed liquor + 500 mL 0.4 N Na2CO3, (3) 2 L mixed liquor + 100 mL 0.2 N NaOH + 400 mL distilled water, (4) 2 L mixed liquor +100 mL 0.8N HCl + 400 mL distilled water. The pH values for sludge samples were 7.4, 10.4, 11.1, and 2.0, respectively.  The sludge samples from the aerobic phase of the ozonated SBR were prepared and completely mixed as: (1) 2 L mixed liquor + 500 mL distilled water, (2) 2 L mixed liquor + 500 mL 0.4 N Na2CO3, (3) 2 L mixed liquor + 100 mL 0.2 N NaOH + 400 mL distilled water, (4) 2 L mixed liquor +100 mL 0.8 N HCl + 400 mL distilled water. The pH values for these sludge samples were 7.2, 10.2, 11.0, and 2.0, respectively. Each sludge sample was then treated by the plug-flow reactor with and without ozone injection. Table 1 lists types, compositions and characteristics of sludge for ozone treatment.  Table 2.3  Types, composition and characteristics of sludge for ozone treatment 67 Types of Sludge Sludge prepared for treatment by the plug-flow reactor Solids concentration TSS(VSS) (mg/L) VSS to TSS ratio (%) Particulate Size  by Volume Mixed liquor from aerobic tank in UBC MEBPR Pilot Plant  2 L no dilution 11,580 (8,280) 71.5 D(0.1)=10.6 µm, D(0.5)=23.0 µm, D(0.9)=116.6 µm 2 L 75% diluted by effluent 8,740 (6,300) 72.1 2 L 50% diluted by effluent 5,700 (4,300) 75.4 Mixed liquor from aerobic phase of the control SBR  (SRT = 50 d) 2 L mixed liquor + 500 mL distilled water 4,920 (4,100) 83.3 D(0.1)=33.7 µm D(0.5)=128.1 µm D(0.9)=339.8 µm 2 L mixed liquor + 500 mL 0.4 N Na2CO3 2 L mixed liquor + 100 mL 0.2 N NaOH + 400 mL distilled water 2 L mixed liquor +100 mL 0.8 N HCl + 400 mL distilled water Mixed liquor from aerobic phase of the SBR combined with sludge ozonation (SRT = 50 d) 2 L mixed + 500 mL distilled water 4,613 (3,680) 79.8 D(0.1)=31.9 µm D(0.5)=87.0 µm D(0.9)=181.3 µm 2 L mixed liquor + 500 mL 0.4 N NaHCO3 2 L mixed liquor + 100 mL 0.2 N NaOH + 400 mL distilled water 2 L mixed liquor +100 mL 0.8 N HCl + 400 mL distilled water  For each trial, about 2 L sludge was treated in a once-through way by the plug-flow reactor. Sludge was sampled before and immediately after treatment. Triplicate samples were taken from each sludge for mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) measurement. The soluble constituents were measured after centrifugation of mixed liquor samples (24,000 rcf for 15 minutes) and filtration of the centrate through 0.45 μm filters. The sludge samples were 68 analyzed on the same day as the batch tests. The soluble constituent samples were stored at 4 o C until the analyses were performed in the Environmental Engineering Laboratory at UBC. The parameters, such as pH, COD, TSS, VSS, particle size distribution and adenosine triphosphate (ATP), were analyzed.  2.4 Analytic Methods 2.4.1 Sampling plan Samples of influent wastewater and the process effluent discharged as membrane permeate were collected by grab sampling daily. The concentrations of soluble constituents were measured in filtrates collected after filtration through 0.45 μm filters. The total COD, ammonium-nitrogen (NH4-N), nitrate-nitrogen (NO3-N) and nitrite-nitrogen (NO2-N), and orthophosphate (PO4-P) concentrations were analyzed. During phosphorus batch tests, the concentration of the volatile fatty acids (VFA) in mixed liquor was measured. The samples were preserved immediately after collection and/or filtration. The samples were stored at 4 ℃ until the analyses were performed at the Environmental Engineering Laboratory at UBC. Mixed liquor samples were collected during the aerobic stage from each of the SBRs daily and  were analyzed for total and volatile suspended solid content (TSS/VSS). Duplicate sample analysis was conducted. The samples were also filtered through glass micro-fibre filter paper to separate soluble fractions from particulate fractions. Mixed liquor samples were also collected at random for particle size distribution measurement and for measurement of adenosine triphosphate (ATP) for measuring microorganism activity. The sample preparation procedures are summarized in Table 2.4.  69 Table 2.4  Sample preparation protocols  2.4.2 Analytical Methods 2.4.2.1 Total and Volatile Suspended Solids (TSS/VSS) TSS and VSS were measured according to Standard Methods (APHA et al., 2012; Rice et al., 2012). About 5 mL volumes of well-mixed samples were filtered through glass micro-fibre filter paper (Whatman TM934 AHTM) and the residues retained on the filter papers in the aluminum dish were dried at least 2 hours at 105 ℃ before being cooled and weighed. The filter papers and dried residues were transferred to a muffle furnace at 550 ℃, heated for an hour and then cooled and weighed again. TSS and VSS were calculated using the following equation, TSS (mg/L)  = (X − Y) × 106/𝑉                (2-3) VSS (mg/L)  = (X − Z) × 106/𝑉                (2-4) Where, X = Mass of the dish, filter paper and filtered sludge after drying at 105 ℃, mg, Y = Tare mass of the aluminum dish containing glass fiber filter, mg, and Parameter Sampling method Sampling frequency Preservation Holding time (4 C) COD Grab, 2 mL Daily - < 28 days NH4+-N Grab, 6-8 mL Daily 2 drops of 5% v/v H2SO4 < 28 days PO4- P Grab, 6-8 mL Daily 2 drops of 0.10% phenyl mercuric acetate < 28 days NO3-N NO2-N Grab, 6-8 mL Daily  2 drops of 0.10% phenyl mercuric acetate < 28 days TSS VSS Grab,  20~40 mL Daily - < 14 days VFA Grab, 2 mL Random A drop of 10% H3PO4 < 14 days 70 Z = Mass of the dish, filter paper and residual filtered material after ignition at 550 ℃, mg.  2.4.2.2 Chemical Oxygen Demand (COD) The closed reflux colorimetric method was used for determining COD as described in Standard Methods 5220 D (APHA et al., 2012). Samples were added to a previously made reagent which was prepared by mixing 2.7 mL of sulfuric acid with 1.2 mL of digestion solution. The digestion solution was made by adding 10.216 g of K2Cr2O7, 167 mL of H2SO4, and 33.3g HgSO4 in 1 L of distilled water for high range of COD. For low range (0-200 mg/L) COD 1.022 g of K2Cr2O7 was used. Low range reagents were used for the samples of the effluent from the membrane permeate and high range reagents were used for the samples of wastewater influent. The samples were then analyzed by a HACH DR2800TM portable spectrophotometer.  2.4.2.3 Ammonium Nitrogen Ammonium nitrogen was analyzed by using the automated phenate method according to Standard Methods 4500-NH3 G(APHA et al., 2012). A Lachat Quik-Chem® 8000 flow injection analyzer (Milwaukee, WI, USA) was used for the analysis.   2.4.2.4 Nitrate and Nitrite Nitrogen Samples were analyzed according to the cadmium reduction flow injection method described in Standard Method 4500-NO3 I- (APHA et al., 2012) by using the Lachat Quik-Chem® 8000 flow injection analyzer. 71 2.4.2.5 Ortho-Phosphate Phosphorus Two drops of 0.10% phenyl mercuric acetate was added as the preservative for ortho-phosphate. Ortho-phosphate phosphorus was determined according to the Standard Method 4500 P G (APHA et al., 2012) by using the Lachat Quik-Chem® 8000 flow injection analyzer.  2.4.2.6 Volatile Fatty Acids The volatile fatty acids (VFA) were measured by using gas chromatography (GC), with a Hewlett Packard (Palo Alto, CA, USA) 5890 Series II gas chromatograph, equipped with a flame ionization detector (FID).  2.4.2.7 pH and DO The DO concentrations and pH values were measured with a portable DO meter (Hach® HQ30d).   2.4.2.8 Sludge ATP The sludge ATP concentration was measured by the Luminultra® QG21W test kits provided by LuminUltra Technologies Ltd., Fredericton, New Brunswick, Canada.  2.4.2.9 Microscopic Observation The microscopic images of sludge flocs were obtained with a phase contrast microscope, made by Eclipse E, Nikon Instrument, Melville, NY, USA. 72 2.4.2.10 Particle Size Distribution Particle size distribution was measured by a Malvern Instrument (Westborough, MA, USA) Mastersizer 2000 analyzer, with a Hydro S (Malvern) automated sample dispenser unit. Material sizes ranged from 0.2 μm to 2000 μm.  2.4.2.11 Sludge Volume Index (SVI) and Zone Settling Velocity (ZSV)  The sludge volume index (SVI) and zone settling velocity (ZSV) were measured in a 1-L cylinder according Standard Method 2710D and 2710 E (APHA et al., 2012).  2.4.2.12 16S Ribosomal RNA Sequencing Two independent sludge samples were taken from each SBRs at SRT = 50 days and sent out to Microbiome Insights Inc. (Vancouver, BC) for 16S rRNA gene analysis. The 16S rRNA gene was sequenced via V4 amplicons generated from mouse gut samples on a MiSeq. MiSeq-generated Fastq files were quality-filtered and clustered into 97% similarity operational taxonomic units (OTUs) using the mothur software package.  2.5 Batch Test Methods 2.5.1 The Nitrification Rate To measure nitrification rates, batch kinetic tests were conducted in the SBR tank after the 2nd aeration stage and before the settling stage, following 1 hour of anoxic mixing. The dissolved oxygen concentration, temperature and pH were monitored during the tests.  73 At time zero, 500 mL of 0.3 N NH4Cl solution and 500 mL 0.3 N NaHCO3 pH buffer solution were added to each SBR (total volume was about 20 L) to establish an initial NH4-N concentration of roughly 105 mg-N/L. The mixed liquor was aerated at the same time. After 1 to 2 minutes following substrate addition, a sample was collected from each reactor, to measure the initial concentration of ammonium-nitrogen (NH4-N) and nitrite + nitrate-nitrogen (NOX-N). Samples of mixed liquor were then taken every 15 minutes over the total sampling time of 120 minutes. Mixed liquor samples were centrifuged for 3-5 minutes at 550 relative centrifugal force, and the supernatant was passed through a 0.45-µm-pore-size filter to obtain 10 mL of soluble sample for subsequent measurements.  Triplicate mixed liquor samples for total suspended solids (TSS) and volatile suspended solids (VSS) were also collected from each reactor at the beginning and end of the experiment, and the six values were averaged to obtain the suspended solids concentration of the batch test.  2.5.2 The Phosphorus Removal Rate The phosphorus release and uptake rates were measured in the SBR after the 2nd aeration stage and before the settling stage, following about 4 hours under anoxic/anaerobic conditions to eliminate any nitrates present in the mixed liquor. The dissolved oxygen concentration, temperature and pH were monitoring during the test. Subsequently, the mixed liquor was aerated for about 1 h to allow uptake of the orthophosphates that were released to the solution due to endogenous activity of phosphorus-accumulating organisms and overall biomass decay.  At time zero, the mixing impellers were turned on and a 300 mL of 0.2 N sodium acetate solution was added to each reactor to establish an initial acetate concentration of roughly 55 74 mg/L. After 1~2 min from substrate addition, a sample was collected from each reactor to measure the initial concentration of acetate and orthophosphate PO4–P. Anaerobic conditions were maintained for 120 min and samples were collected every 15 min until the completion of this phase. The maximum specific P-release rate and the acetate uptake rate were calculated for the first 45 min of the experiment using linear regression analysis.  At time 120 min, aeration was re-started and samples for acetate and PO4–P were collected every 15 min during the 120 minutes of the batch aerobic phase. The maximum specific aerobic P uptake rate was calculated for the first 45 min of straight slope. During the test, the dissolved oxygen concentration, temperature and pH were monitored. Triplicate mixed liquor samples for TSS and VSS were collected from both reactors at the beginning and end of the experiment, and the six values were averaged to obtain the TSS and VSS concentration.  2.5.3 Heterotrophic Decay Rate The batch respirometric method (Ekama et al., 1986) was used to determine the decay coefficient outlined in the ASM1 documentation (Henze et al., 1987). About 4 L mixed liquor was taken from the SBR tank and then placed into two 2-L flasks which served as respirometers. An air diffuser was placed in the bottom of each flask and the air flow rates were adjusted to maintain the dissolved oxygen (DO) concentration at 4 ~ 6 mg O2/L during the tests. Magnetic stir bars and a mixing plate were used to keep the mixed liquor in suspension. The mixed liquor samples were tested in duplicate. The mixed liquor was continuously stirred and aerated.  75 The oxygen uptake rate (OUR) was measured by stopping the aeration and monitoring the decreasing dissolved oxygen concentration in the respirometer. An OUR measurement was conducted in this manner twice a day for a period of 7 to 10 d.  The pH of the respirometer was maintained at around 7 and nitrification was inhibited by adding 0.16 g nitrification inhibitor formula 2533 (HACH Company) per 300 mL of sample. The foam generated in the upper part of the respirometers was sprayed by distilled water daily to keep bacteria in liquid suspension. Evaporative losses of water were also compensated for by the addition of distilled water. The traditional decay coefficient, bH was determined by the slope of a plot of the natural logarithm of the OUR versus time.  The temperature was compensated for by correction to a reference temperature of 20ºC by equation, bH  =  bH,20℃  ×  θ(T – 20)                          (2-5) Where, bH = decay coefficient at temperature T, ℃, d-1, bH, 20℃ = decay coefficient at 20ºC, d-1, θ = temperature acitivity coefficient, 1.04 (Tchobanoglous et al., 2003), and T = temperature, ℃.  2.5.4 The Specific Growth Rate (μH) The growth rate of heterotrophic microorganisms (μH) was determined using the batch respirometric method (Kappeler and Gujer, 1992). A sample of 2 L primary effluent of wastewater from the UBC pilot plant was centrifuged for 15 minutes and the centrate was used 76 as the substrate for measurement. The mixed liquor samples were taken from the SBR tank and tested. Again, 2-L flasks were used as respirometers and an air diffuser was placed in the bottom of each flask.  Magnetic mixers with stirring bars were used to keep the respirometer contents in suspension.  About 10 mL mixed liquor from the SBR was added to each flask as the seed and the wastewater and biomass were mixed in a COD to VSS ratio of about 4:1. Nitrification was inhibited by the Hach formula 2533 described above. The contents of the flasks were continuously stirred and aerated. The OUR was measured immediately after mixing until a sharp decrease in rate was observed. The specific growth rate was determined from the slope of the natural logarithm of the OUR versus time plot according to equation 2-6 ln (� 𝑟𝑂2(𝑡)𝑟𝑂2(𝑡0)� = (𝜇𝐻 − 𝑏𝐻)              (2-6) where,  𝑟𝑂2(𝑡0) = the oxygen utilization rate at the beginning of the test, mg/L/d,  𝑟𝑂2(𝑡) = the oxygen utilization rate at time t when maximum rate was reached, mg/L/d, 𝜇𝐻 = the specific growth rate, d-1, and 𝑏𝐻 = the decay rate, d-1. The measured rates were corrected to a reference temperature of 20ºC by equation 2-7 as follows, µH  =  µH,20℃  ×  θ(T – 20)                          (2-7) Where, 77 µH = growth rate at temperature T ℃, d-1, µH, 20℃ = growth rate at 20ºC, d-1, θ = temperature activity coefficient, 1.094 (Tchobanoglous et al., 2003), and T = temperature, ℃.  2.5.5 The Maximum Growth Rate and the Half Velocity Coefficient According to the Monod Equation (Monod 1949), the growth rate of microorganisms can be expressed  by, µ = 𝜇𝑚 𝑆𝐾𝑠+𝑆              (2-8) Where, µ = the growth rate at substrate concentration S (mg/L), d-1,  µm = the maximum growth rate, d-1, S = the concentration of the substrate, mg/L, and, Ks = the half velocity coefficient, mg/L. The equation 2-8 can be converted to Equation 2-9 as, 1𝜇= 1𝜇𝑚�𝐾𝑠+𝑆𝑆� = �𝐾𝑠𝜇𝑚�1𝑆+ 1𝜇𝑚             (2-9) From the Equation 2-9, the variables 1𝜇 and 1𝑆 can be modeled by a linear relationship. The values of the terms  𝐾𝑠𝜇𝑚 and µm can be determined from the slope and the intercept of a plot of 1𝑆  versus 1𝜇. 78 Therefore, the maximum growth rate (µm) and the half velocity coefficient (Ks) for heterotrophic microorganisms can be calculated.  The growth rate of heterotrophic microorganisms (μ) at different substrate concentrations was determined using the batch respirometric method described above. Four different concentrations of substrate were used for the test. Primary effluent from the UBC pilot plant was centrifuged at 24900 rcf for 15 minutes using Thermo IECTM multi centrifuge and the centrate was used as the highest concentration of substrate. The centrate was diluted with distilled water to 85%, 70% and 55% of the original concentration to serve as the other three test concentrations of substrate. About 20 mL mixed liquor samples were taken from the SBR tank and completely mixed. 5 mL mixed liquor was added to each flask as the seed at the same time. Nitrification was inhibited by the Hach formula 2533 described above. The contents of the four flasks were continuously stirred and aerated. The OUR was measured immediately after mixing the contents of each flask until a sharp decrease in rate was observed. The specific growth rate was determined from the slope of the natural logarithm of the OUR versus time plot according to Equation 2-6.   79 Chapter 3:  Results of Sludge Reduction by Mixed Liquor Ozonation  3.1 The Operating Conditions  The comparative experiment started on Jun 26th, 2015 and ended on April 28th, 2016. There were two independent experimental phases (Phase I SRT = 25 days and Phase II SRT = 50 days). The operating conditions were described in Chapter 2. The pH and DO profiles for the two SBRs were different, as illustrated in Figure 3.1. For both reactors, the pH increased during the anoxic stage due to denitrification and then slightly decreased during the anaerobic stage after denitrification was complete. The pH further decreased in the 1st aerobic stage due to the alkalinity requirement for nitrification. There was usually a pH increase during the 2nd aerobic stage as CO2 in the mixed liquor was stripped by aeration (Cohen and Kirchmann 2004) following the completion of the nitrification reaction. 80   Figure 3.1  The DO and pH profiles for the SBRs (top: the control SBR, bottom: the ozone treated SBR)  The major difference between the control reactor and the ozone-treated reactor was that the DO concentration exhibited a sharp peak during the mixed liquor ozone treatment. Because pure 81 oxygen was used to generate ozone and the maximum ozone concentration achieved was only 10%, high concentration oxygen gas was released during mixed liquor ozonation and the DO concentration dramatically increased after even a short ozone treatment. The pH of the mixed liquor after ozone treatment decreased slightly, which was due to ozone decomposition and alkalinity consumption for nitrifying the inorganic ammonium released by the mixed liquor ozonation.   3.2 The Inventory Changes and the Ozone Dosages 3.2.1 The Suspended Solids Inventory Changes during the Experiment The mass of total suspended solids in both reactors during the 311 days of experimentation is illustrated in Figure 3.2. The inventory increased after the SRT change: the total mass of TSS was 40 ~ 80 g at SRT = 25 d and 80 ~ 120 g at SRT = 50 d.  The total mass of TSS in the ozone-treated reactor was significantly lower than that in the control reactor with time during operation at SRT = 25 d. At SRT=50 d, a reduced inventory for the ozonated reactor was also measured. However, when the substrates were switched at the beginning of Phase II at SRT = 50 d, sludge bulking occurred in the control SBR, which caused significant sludge loss and the total mass of TSS dropped sharply. No sludge bulking was observed in the ozonated SBR, although the TSS inventory in the ozonated SBR decreased, too. In general, the suspended solids inventory in the ozonated reactor was lower than that in the control reactor. 82  Figure 3.2  The total mass of TSS (the inventory) in both SBRs during the 311 days of experimental operation.  3.2.2 The Ozone Dosages The ozone dosage is the dominant factor that controls the performance of sludge ozone treatment. The values of ozone dosage (defined as the mass of ozone applied in the reactor per mass of TSS in the sludge) reported in many literature studies (Foladori et al., 2010; Manterola et al., 2008; Yan et al., 2009) range from 0.01 g O3/g TSStreated up to 1 g O3/g TSStreated. To save energy, a low dosage is preferred for sludge ozone treatment. In the present study, the ozone was introduced by a Venturi injector and the mixed liquor ozone-treatment reactor was a semi-batch system, the time factor needs to be considered during dosage calculation. 83 • The ozone dosages and the VSS/TSS ratios In the present study, the gas (ozone and oxygen) flow rate was about 0.3 - 0.4 L/min and the liquid flow rate was about 3 L/min. The ozone treatment time was about 1 minute per cycle and in each cycle 80-90 mg ozone was introduced to the Venturi injector reactor. About 320 - 360 mg ozone was applied to the reactor per day since the cycle time of SBRs was 6 hours and there were 4 cycles every day. The ozone was assumed to be completely transferred and consumed by the mixed liquor in each cycle since: (1) ozone was fast transferred to and consumed by the mixed liquor in the ozone-treatment reactor; (2) any remaining ozone was mixed with and consumed by the mixed liquor in the SBR, although a small amount of ozone escaped to the headspace of the covered SBR. The ozone dosages were calculated by following equation, D = 𝑚×𝑆𝑅𝑇𝑉×𝑋�   (3.1) Where, m = the masss of ozone consumed per day, g O3/d; SRT = the nominal sludge retention time, d; V = the total volume of the SBR, L; and, X� = the average concentration of the mixed liquor suspended solids in the SBR, g TSS /L. Figure 3.3 shows the overall ozone mass applied per day and the calculated dosage (g O3/g TSS) and the ratio of VSS and TSS. The average dosages were 0.20 g O3/g TSS at SRT = 25 d and 0.18 g O3/g TSS at SRT=50 d. Because the solids concentration varied during the experiment, the dosage ranged from 0.006 to 0.022 g O3/g TSStreated per cycle (the mass of ozone applied in 84 the ozone reactor per mass of TSS in the mixed liquor for each SBR cycle). At SRT = 25 d, the ozone dosage was generally higher with 0.012 - 0.022 g O3/g TSStreated per cycle due to the lower MLSS concentration; At SRT = 50 d, the dosage was lower at 0.006 - 0.010 g O3/g TSStreated per cycle due to the higher MLSS concentration.  Figure 3.3  The ozone dosage and VSS/TSS ratio for 311 days The VSS/TSS ratio roughly indicates the organic content of sludge. The observed VSS/TSS changed with SRT and ozone dosage. At SRT = 25 d, the VSS/TSS ratio was about 85% for the control and about 80% for the ozone-treated system, which indicated that ozone treatment may cause the organic content to decrease in the mixed liquor. The results of the t-tests with significance level of 0.05 indicated that there was a statistically significant difference in the VSS/TSS ratios of the two systems. However, at SRT = 50 d, there was no significant difference 85 in the VSS/TSS ratio between sludge from the control system and from the ozonated system. The VSS/TSS ratios in both reactors varied between 80 - 85% depending on the F/M ratios: the higher the F/M ratio, then the higher the observed VSS/TSS ratio. At SRT = 50 d, the average MLSS concentrations from both reactors were high and therefore, the relative ozone dosage was low. The similar VSS/TSS ratios under these conditions may indicate that stabilization of organic material was similar in both systems at the longer SRT, perhaps because the ozone dose was lower than that at the 25 d SRT.  • Calculations of ozone dosages by other ways The ozone dosage can be calculated in various ways. The ozone dosage can be calculated by the mass of ozone consumed divided by the mass of mixed liquor treated in the Venturi reactor, which is similar to Equation 1-18. The ozone dosage can also be defined as the mass of ozone consumed per mass of excess sludge generated wasted in the control reactor. It also can be defined as the mass of ozone applied per mass of total COD removed in the system. Figure 3.4 shows a comparison of ozone dosages calculated by three different approaches. The ozone dosages by the mass of excess sludge generated in the control were generally high and the dosages by the mass of the mixed liquor treated were low among all dosage calculations. 86  Figure 3.4  The comparison of ozone dosages by different calculations  3.2.3 Substrate Changes During Mixed Liquor Ozonation The mixed liquor ozone treatment started after the 1st aerobic reaction phase of each SBR cycle and lasted for 1 minute. This was then followed by anaerobic mixing and further aerobic reaction (the 2nd aeration) to degrade the lysate generated by sludge ozonation. Figure 3.5 shows the soluble COD (SCOD), NOx-N, NH4+-N and PO4-P concentrations before and after mixed liquor ozone treatment. In Figure 3.5(A), the significant amount of soluble COD that was released by the short period of mixed liquor ozonation subsequently decreased quickly, presumably by microbial degradation. Ammonium release was also observed during mixed liquor ozonation (Figure 3.5 (C)), after 87 which the ammonium concentration also gradually decreased. There was no significant measureable change in NOx-N concentration during mixed liquor ozonation, but the subsequent increase indicates that the released ammonium was nitrified. The phosphate concentration slightly increased during ozonation and then decreased, which indicated that a small amount of ortho-phosphate was released by mixed liquor ozonation and this was then taken up by PAOs. 88  Figure 3.5  The soluble COD (SCOD), inorganic nitrogen and ortho-phosphate concentrations before and after mixed liquor ozonation 89 3.3 Sludge Yield Reduction 3.3.1 Sludge Yield Reduction at SRT = 25 d The observed sludge yield was calculated by mass balance considerations involving the cumulative suspended solids mass flows in the influent and in wasted sludge, the MLSS inventory in the SBRs, and the cumulative COD removed. The cumulative COD removed and suspended solids masses are illustrated in Figure 3.6 for the control reactor and in Figure 3.7 for the ozonated system. By developing a mass balance for total suspended solids, the yield can be calculated using Equation 3.2 over the total period of operation at SRT=25 d and SRT =50 d, respectively. Y = ∑𝑇𝑆𝑆𝑤𝑎𝑠𝑡𝑒𝑑+∑𝑇𝑆𝑆𝑒𝑓𝑓−∑𝑇𝑆𝑆𝑖𝑛𝑓+(𝐼𝑁𝑉𝑖−𝐼𝑁𝑉0)∑𝐶𝑂𝐷𝑟𝑒𝑚𝑜𝑣𝑒𝑑  (3-2) Where, Y = the yield, g TSS/g COD; 𝑇𝑆𝑆𝑤𝑎𝑠𝑡𝑒𝑑 = total suspended solids wasted, g, 𝑇𝑆𝑆𝑒𝑓𝑓 = total suspended solids in the effluent, g, 𝑇𝑆𝑆𝑖𝑛𝑓 = total suspended solids in the influent, g, 𝐼𝑁𝑉𝑖 = Inventory (the total TSS in the reactor) on day i, g, 𝐼𝑁𝑉𝑖 = Inventory (the total TSS in the reactor) on day 0, g, and 𝐶𝑂𝐷𝑟𝑒𝑚𝑜𝑣𝑒𝑑 = the total influent COD minus total effluent COD, g. 90  Figure 3.6  The cumulative COD removal  and suspended solids mass balance data for the control reactor at SRT = 25 d   Figure 3.7  The cumulative COD removal  and suspended solids mass balance data for the ozonated system at SRT=25 d  91 The suspended solids concentration in the effluent was assumed to be zero because the ultrafiltration membrane was used to polish effluent before discharge and the suspended solids retained in the MBR tanks were returned to the main SBRs manually.  The sludge yields for both reactors were then calculated from the data shown in Figure 3.6 and Figure 3.7 by Equation 3-2. Figure 3.8 shows the yield comparison between the control and the ozone-treated systems at SRT = 25 d.   Figure 3.8  The observed sludge  yield comparison between the control and the ozonated reactors at SRT = 25 d.  The yield was decreased by 35% in the ozonated reactor compared to that in the control reactor for SRT = 25 d. The sludge yield of the ozonated reactor was 0.177 g TSS/ g CODremoved (0.145 g VSS/ g CODremoved) and that of the control was 0.273 g TSS/ g CODremoved (0.224 g VSS/ g CODremoved). The sludge reduction percentage is comparable to the results reported in related literature studies (Cui and Jahng, 2004; Gardoni, 2015; Saktaywin et al., 2005), in which the recycled sludge, rather than mixed liquor, was treated by ozone.  92 3.3.2 Sludge Reduction at SRT = 50 d In the same way, the cumulative suspended solids in the influent and in wasted sludge, MLSS inventory in the SBRs, and the cumulative COD removed were calculated and the results are illustrated in Figure 3.9 for the control reactor and in Figure 3.10 for the ozonated reactor, both of which were operated at a 50 d SRT. The yields for both reactors were calculated by using the data (from October 18, 2015 to April 28, 2016) shown in Figure 3.9 and Figure 3.10 by Equation 3.1. The TSS in the effluent was negligible due to membrane filtration. Figure 3.11 shows the yield comparison between the control and the ozone-treated systems at SRT = 50 d. Figure 3.11 indicates that the yield was decreased by 42% in the ozonated reactor compared to that in the control at SRT = 50 d. The sludge yield of the ozonated reactor was 0.081 g TSS/ g CODremoved (0.064 g VSS/ g CODremoved) and that of the control was 0.047 g TSS/ g CODremoved. (0.036 g VSS/ g CODremoved). At SRT = 50 d, the operating F/M ratios were low, at 0.09 ~ 0.13 g COD/ g VSS∙d for both reactors. In the absence of an external carbon source, the cellular tissues, intracellular reserve materials, and decayed/dead cells are used for maintenance requirements of bacteria (Van Loosdrecht and Henze, 1999) or endogenous metabolism. At a long SRT with low F/M ratios, it was assumed that most of available energy was required for maintenance functions, and the amount of energy available for growth of biomass decreased (Low and Chase, 1999). Therefore, a significant reduction in sludge yield was achieved at SRT = 50 d.   93  Figure 3.9  The cumulative COD removal  and suspended solids mass balance data for the control reactor at SRT = 50 d   Figure 3.10  The cumulative COD removal  and suspended solids mass balance data for the ozonated reactor at SRT = 50 d  94  Figure 3.11  The observed yield comparison between the control and the ozonated reactors at SRT=50 d  3.3.3 Sludge Reduction by Combination of Extended SRT and Mixed Liquor Ozonation An observed sludge yield comparison for the two SRT experiments is illustrated in Figure 3.12.  Figure 3.12  The observed sludge yield comparison between the control and the ozonated reactors during experiment. 95 The operation described here at a 25 d SRT is representative of many full-scale EBPR wastewater treatment plants at the upper range of SRT (Tchobanoglous et al., 2003). In the present experiment, the observed sludge yield was 0.273 g TSS/ g CODremoved (0.224 g VSS/ g CODremoved) when the F/M ratios were 0.16 ~ 0.22 g COD/ g VSS∙d. If combined with mixed liquor ozone treatment at a longer SRT (50 d), an observed sludge yield of 0.047 g TSS/ g CODremoved (0.036 g VSS/ g CODremoved) was achieved, which is equivalent to about an 80% reduction in sludge production. Table 3.1 shows a comparison of sludge reduction achieved by different technologies in literature reports.  Table 3.1  Comparison of sludge reduction by different technology Technology for sludge reduction Sludge yield reduction  Reference Enzymatic hydrolysis 39.7% - 68.43 VSS Yang et al., 2010 High pressure homogenisers 20 - 24% TSS Camacho et al., 2002 Ultrasonic disintegration 25 - 90%  TSS Neis et al., 2008;  Zhang et al., 2007; Romero-Pareja et al., 2017 Thermal treatment <55% TSS Camacho et al., 2005 Extended aeration <30% TSS Mahmood and Elliott, 2006 Microbial predation 10% - 23.7% Guo et al., 2007; Zhu et al., 2016 Peroxidation <50% TSS Paul et al., 2006 Chlorination <65% TSS Saby et al., 2002 Fenton 96% TSS He and Wei, 2010 Ozonation  30-100% TSS (secondary wastewater treatment process) Kamiya and Hirotsuji, 1998; Lee et al., 2005; Sakai et al., 1997; Yasui et al., 1996;  Gardoni et al., 2011;  Romero et al., 2015 Long SRT and Mixed liquor ozonation 80% TSS (EBPR system) Present study 96 It seems that ultrasonic disintegration and ozonation are the two most effective sludge reduction options. In the present study, a combination of long SRT and mixed liquor ozonation reduced the sludge yield by 80%, which was high compared with the other technologies reported in the literature.  3.4 The Effluent Quality 3.4.1 Chemical Oxygen Demand (COD) Figure 3.13 shows the measured COD concentrations in samples collected during the experiment.   Figure 3.13  The COD concentrations for the influent and the effluent of both reactors The effluent quality with ozone treatment was not affected and there was no statistically significant difference of effluent COD concentrations between the two systems as judged by t-97 tests at a significance level of 0.05. Both the control and the ozone-treated systems achieved about 90 ± 1% COD removal during experiment phases.   3.4.2 Inorganic Nitrogen  Figure 3.14 shows the ammonium-N and nitrate+nitrite-N (NOx-N) concentrations in the influent and effluent for both reactors. The effluent concentrations of ammonium in both rectors were similar with an averaged 99 ± 1 % removal efficiency. There was no statistically significant difference in ammonium removal efficiencies between the two systems detected by t-tests at a significance level of 0.05, which indicates that the mixed liquor ozone treatment did not affect nitrification.  At SRT = 25 d, the NOx-N concentration in the effluent was generally higher for the ozonated reactor than for the control. The inorganic nitrogen removal efficiencies were 92 ± 1% for the control reactor and 83 ± 1% for the ozonated system. The lower inorganic nitrogen removal for the ozonated reactor may have beeen caused by the nitrogen released by sludge ozone treatment.  At SRT =50 d, both reactors achieved 79 ± 1% inorganic nitrogen removal efficiency, and there was no statistically significant difference in inorganic nitrogen removal efficiencies between the two systems as determined by t-tests at a significance level of 0.05. 98  Figure 3.14  The inorganic nitrogen concentrations for both reactors  3.4.3  Ortho-phosphorus Concentrations Figure 3.15 shows the measured ortho-phosphate concentrations in influent and effluent samples taken during the experiment.  At SRT = 25 d, the ortho-phosphate concentration in the effluent was generally higher for the ozonated reactor than for the control with a statistically significant difference. The ortho-phosphate removal efficiencies were 95 ± 1% for the control reactor and 92 ± 2% for the ozonated system. The lower ortho-P removal for the ozonated reactor may be due to the high ozone dosage applied, which caused low MLSS and PAOs inventories in the ozonated reactor. 99 At SRT = 50d, the orhto-phosphate concentrations in effluent from the ozonated reactor were comparable to those in the control reactor effluent, which indicated that P removal was not significantly affected by mixed liquor ozonation. Although several P removal upsets occurred due to air compressor failures and disc diffuser fouling during the experiment, overall about 94 ± 1% ortho-P removal efficiency was achieved in both systems.   Figure 3.15  The ortho-phosphorus concentrations for both SBRs  3.4.4 Summary The Appendix A shows all the COD, inorganic nitrogen and ortho-phosphorus concentrations in the influent and effluent for both reactors during this comparative study.  100 Both the control and the ozone-treated systems achieved about 90 ± 1% COD removal and 99 ± 1 % ammonium removal efficiencies with no statistically significant difference during the experiment.  At SRT = 25 d, the inorganic nitrogen and the ortho-phosphate removal efficiencies were lower (83 ± 1% and 92 ± 2%) for the ozonated system than those (92 ± 1% and 95 ± 1%) for the control reactor.  At SRT =50 d, the effluent quality from the ozone-treated SBR system was comparable to that from the control and both reactors achieved 79 ± 1% inorganic nitrogen removal and 94 ± 1% ortho-P removal efficiencies. This indicated that low-dosage mixed liquor ozone treatment applied to an EBPR process at long SRT may not affect the effluent quality.   3.5 The Characteristics of the Mixed Liquor 3.5.1 The Settling Characteristics Figure 3.16 shows sludge volume index (SVI) for sludge from the control system and the ozonated system at SRT = 25 d and SRT = 50 d. From the figure, The SVIs were 81 ± 30 and 63 ± 5 mL/g at SRT = 25 d and SRT = 50 d respectively for the sludge in the ozonated reactor, and these were significantly lower than those of the control sludge with 226 ± 12 mL/g at SRT = 25 d and 136 ± 12 at SRT = 50 d. The settling properties of the mixed liquor in the ozonated reactor were greatly improved, which was consistent with results reported by other researchers (Deleris et al., 2002; Nilsson et al., 2014; Paul and Debellefontaine, 2007). The good settling properties 101 of the mixed liquor in the ozonated reactor also improved the stability of effluent quality which was described above at 3.4.  Figure 3.16  The SVI of mixed liquor from the control and the ozonated reactor at different SRTs Figure 3.17 shows the interface height with time for mixed liquor from the two reactors during settling tests. The zone settling velocity (ZSV) can be determined by estimating the slope of the first linear part of each curve in Figure 3.16. The ZSV of mixed liquor from the ozonated reactor was 4-8 times higher than that from the control reactor. The low SVI and high ZSV of mixed liquor from the ozonated reactor indicated improved sludge settling properties at both SRT = 25 d and SRT = 50 d. The improved sludge settling with mixed liquor ozonation suggests that high sludge concentrations could be maintained, even in conventional activated sludge systems with secondary clarification.  102  Figure 3.17  The zone settling velocity test for the mixed liquors from the control and the ozonated reactors  In a conventional activated sludge process with a secondary clarifier, the potential for operation at an elevated SRT is limited because of the accompanying increase in mixed liquor suspended solids concentration. The recently developed MBR and granular sludge processes can help to overcome this limitation. From the results of this experiment, mixed liquor ozonation can be an alternative method to increase SRT and sludge solids concentration in conventional activated sludge system. In addition, sludge production may be reduced with ozonation.  103 3.5.2 Particle Size Distribution It was assumed that mixed liquor particle size distribution would be affected by ozonation, since ozonation has been reported previously to result in the formation of smaller flocs (Park et al., 2003; Zhao et al., 2007). The particle size distributions for the mixed liquors from both the control and ozonated reactors were measured every two weeks during the present study. Typical particle size distributions are illustrated in Figure 3.18, Figure 3.19 and Figure 3.20. The results show that particle size distribution in mixed liquor from the ozonated reactor changed significantly compared with that from the control. The particle size distribution by volume (Figure 3.18) shows that the median particle size of mixed liquor from the ozonated reactor was smaller than that from the control system. In addition, the sharper “bell curve” for the ozonated reactor in Figure 3.18 indicates that the particle size distribution of mixed liquor from the ozonated reactor was more uniform than that from the control.  Figure 3.19 shows a typical particle size distribution by number for mixed liquor from both reactors. It shows that the size of most particles in mixed liquor was between 2 and 20 µm, a size range that would include free bacteria and small flocs (Wu et al., 2009). For mixed liquor from the control reactor, the distribution was sharp and the median size of particles was 3 - 4 µm, which indicated that free bacteria may have composed the fine particles in the control system. For mixed liquor from the ozonated reactor, the median size of particles was reduced to 2 - 3 µm, which may have been the result of cell lysis by ozonation such that more fine particles were generated. 104 Figure 3.20 shows a typical particle size distribution by surface area for sludge from both reactors. The median surface area of particles in sludge from the ozonated reactor (d(0.5)=40-50 µm) was less than that from the control (d(0.5)=60 - 70 µm).  It has been reported that the particle size distribution in mixed liquor did not change significantly after low dosage ozone treatment at lower dosages of 25 - 50 mgO3/g DS(solid concentration) (Zhang et al., 2009). Ozonation may reduce the numbers of small flocs and increase those of medium flocs (2 – 20 μm), which would be similar to results reported by other researchers(Bougrier et al., 2006).   Figure 3.18  A typical particle size distribution by volume for mixed liquor from both systems 105   Figure 3.19   A typical particle size distribution by number for mixed liquor samples from the  two experimental systems   Figure 3.20  A typical particle size distribution by surface area for mixed liquor samples from the  two experimental systems.  106 3.6 Summary In this comparative study, it was observed that low-dosage mixed liquor ozone treatment applied to an EBPR process may be a feasible way to reduce sludge production. A 35% sludge reduction at SRT =25 d and a 42% reduction at SRT=50 d were achieved without affecting the effluent quality.  The sludge settling properties were also significantly improved compared with the control. This may be due to changes in particle size distribution.    107 Chapter 4: Heterotrophic and Nutrient Removal Kinetics  The mixed liquor ozonation may affect heterotrophic and nutrient removal kinetics. The nutrient removal kinetics is associated with the performance (effluent quality) of EBPR system. This chapter describes the comparative batch tests conducted to estimate the kinetics of nitrification and phosphorus removal. The heterotroph kinetic parameters such as decay coefficients, the maximum specific growth rates and half-velocity constants for both reactors were also measured.  4.1 The Nitrogen Removal Rate The results of two independent batch tests of nitrogen removal at SRT = 25 d are shown in Figure 4.1. Although the volumetric nitrification rates of the mixed liquor from the ozonated reactor were similar to or slightly lower than those of the control, the specific nitrification rates in the ozonated system were higher than those of sludge in the control system (Table 4.1). The specific ammonium removal rates were 4.21 - 4.71 g NH4+-N /g VSS∙hr for the control and 5.41 - 6.73 g NH4+-N /g VSS∙hr for the ozonated system. The slightly lower volumetric nitrification rate with ozonation was probably caused by a lower MLVSS concentration and thus a smaller population of nitrifiers.  The results of the nitrification batch tests for the two reactors operated at SRT = 50 d were similar to those at SRT = 25 d (Figure 4.2). The volumetric nitrification rate of mixed liquor in the ozonated reactor was similar to that from the control; and the specific nitrification rate for the ozonated reactor was higher than that for the control. The specific ammonium removal rates 108 were estimated to be 2.77 - 6.02 g NH4+-N /g VSS∙hr for the control and 4.03 - 6.06 g NH4+-N /g VSS∙hr for the ozonated system.  Figure 4.1  The results of nitrification batch tests at SRT = 25 d  Figure 4.2  The results of nitrification batch tests at SRT = 50 d The results are summarized in Table 4.1. The measured specific nitrification rates were adjusted using  an Arrhenius temperature correction coefficient of θ = 1.12 for nitrification (Tchobanoglous et al., 2003). The resulting standardized (corrected by temperature coefficient) nitrification specific rates of sludge in the ozonated reactor were higher than those in the control, which indicated that nitrogen removal was not affected negatively by the sludge ozone treatment in this experiment. This finding is consistent with Sui et al. (2014). The high standardized nitrification 109 specific rate of biomass in the ozonated reactor indicates that, (1) the nitrifiers may be more active in the ozonated system than in the control system; or (2) a larger nitrifier population in the microbial community in the ozonated reactor than that in the control.  Table 4.1  The nitrification specific rate SRT (days)   Nitrification specific rate (mg N/g VSS/hr) Standardized Nitrification specific rate (mg N/g VSS/hr) Temperature (℃)  Control Ozonated Control Ozonated 25 17.2±0.1 3.41 4.86 4.71 6.73 25 19.8±0.1 4.11 5.29 4.21 5.41 50 12.4±0.1 1.15 1.67 2.77 4.03 50 12.9±0.3 2.64 2.66 6.02 6.06  Note: the temperature correction coefficient for nitrification θ = 1.12 (Tchobanoglous et al., 2003)   4.2 The Phosphorus Uptake and Release Rate The results of several phosphorus release and uptake batch tests at SRT = 25 d are shown in Figure 4.3 and the results at SRT=50 d are shown in Figure 4.4. The maximum P-release rates were calculated using data obtained during the first 30 minutes of the tests, when a linear relationship between the ortho-phosphorus concentration and time was observed. The P-uptake rates were calculated from data collected after about 120 minutes under aerobic conditions when VFA had been completely consumed.   110  Figure 4.3  The results of phosphorus release and uptake batch tests at SRT = 25 d  Figure 4.4  The results of phosphorus release and uptake batch tests at SRT = 50 d Table 4.2 shows the specific P-release rates at different reactor operating temperatures and the standardized rates corrected to 20oC. The standardized specific P-release rates were adjusted using a temperature coefficient of 1.078, as proposed by Brdjanovic et al. (Brdjanovic et al., 1997; Brdjanovic et al., 1998). 111 Table 4.2  The specific P-release rate SRT   Temperature (℃) Specific P-release rate (mg P/g VSS/hr) Standardized Specific P-Release rate (mg P/g VSS/hr) Control Ozonated Control Ozonated SRT 25 21.6±0.4 9.08 18.30 8.05 16.22 SRT 25 21.5±0.5 8.00 15.31 7.15 13.68 SRT 50 14.1±0.3 5.39 7.94 8.39 12.37 SRT 50 15.5±0.2 9.80 10.58 13.74 14.84 SRT 50 12±0.3 9.15 10.22 16.68 18.64 Note: the temperature correction coefficient for P-release under anaerobic condition, θ = 1.078 (Brdjanovic et al., 1997; Brdjanovic et al., 1998)  Contrary to conventional wisdom, the results of kinetic batch testing indicated that the maximum P-release rates in the ozonated reactor (13.68 - 16.22 mg P/g VSS h) were significantly higher than those of the control (7.15 - 8.05 mg P/g VSS∙h) at SRT = 25 d. At SRT = 50 d, the specific P-release rate in the ozonated reactor (12.37 - 18.64 mg P/g VSS∙h) was similar to that observed in the control (8.39 - 16.68 mg P/g VSS∙h) with no statistically significant difference detectable by t-tests at alpha level = 0.05. Table 4.3 shows the specific P-uptake rates observed at both SRT = 25 d and SRT = 50 d. The rates were standardized using the temperature correction coefficient of 1.057 proposed by Brdjanovic et al. (Brdjanovic et al., 1997; Brdjanovic et al., 1998). The specific P-uptake rates (12.57 - 13.40 mg P/g VSS∙h) in the ozonated reactor were more than two times those in the control (3.93 - 4.66 mg P/g VSS∙h) at SRT = 25 d. However, at SRT = 50 d, the specific P-uptake rates of the ozonated reactor (3.32 - 6.19 mg P/g VSS∙h) were comparable to those of the control (3.21 - 7.07 mg P/g VSS∙h) with no statistically significant difference detected by t-tests at alpha level = 0.05.   112 Table 4.3  The specific P-uptake rate SRT   Temperature (℃) Specific P-Uptake rate (mg P/g VSS/hr) Standardized  Specific P-Uptake rate (mg P/g VSS/hr) Control Ozonated Control Ozonated SRT 25 21.6±0.4 4.29 13.74 3.93 12.57 SRT 25 21.5±0.5 5.06 14.57 4.66 13.40 SRT 50 14.1±0.3 5.10 4.46 7.07 6.19 SRT 50 15.5±0.2 2.50 2.59 3.21 3.32 SRT 50 12±0.3 4.48 3.79 6.98 5.91 Note: the temperature correction coefficient for P-uptake under aerobic condition, θ = 1.057 (Brdjanovic et al., 1997; Brdjanovic et al., 1998)  The high P-release and P-uptake rates observed in the ozonated reactor in this study were contrary to the results reported by other authors (Gardoni et al., 2011), who observed that the removal efficiency of phosphorus decreased from 70% to as low as 28% as a consequence of reduced biosynthesis after the recycled sludge was ozonated. It is possible that that mixed liquor ozone treatment in the present study had a smaller effect on the PAOs than on other microorganisms because the low ozone dosage was applied over a very short ozone-sludge contact time (1~2 minutes per a 6-hour cycle) compared with the recycle sludge continuous ozonation by other authors (Gardoni et al., 2011).   4.3 The Heterotroph Kinetics To study the solids production during mixed liquor ozonation, the heterotroph kinetic parameters such as the decay rate, and maximum growth rate were measured by batch tests. In this study, the biomass at SRT = 50 d was tested to estimate the batch decay coefficient. The methods used in batch tests were described in Chapter 2.4.3.  113 4.3.1 The Decay Coefficient The endogenous process has a significant impact on the production of waste sludge. Figure 4.5 shows typical results from batch decay tests using MLSS from the control reactor and the ozonated reactor. During the endogenous decay tests, there was no external input of substrate. The only substrates were lysates from the cell decay and associated hydrolysis products. The observed concentration of the biomass and respiration rate decreased slowly and continuously.   Figure 4.5  The results of decay coefficient measurement Table 4.4  The summary of the decay coefficient testing Test Date The control The Ozonated Temperature (℃) Decay coefficient (1/d) Standardized Decay coefficient @20℃ (1/d) R2 Temperature (℃) Decay coefficient (1/d) Standardized Decay coefficient @20℃ (1/d)) R2 3.21 23.4±1 0.165 0.144 0.903 23.4±1.1 0.170 0.149 0.947 3.22 23.2±0.8 0.146 0.129 0.918 23.4±0.6 0.168 0.147 0.889 3.29 23.8±0.6 0.144 0.124 0.950 22.3±0.9 0.199 0.182 0.921 Mean  0.132±0.012  0.159±0.025 Reference: ϴ=1.04 (Tchobanoglous et al., 2003)  114 The decay coefficients measured for the heterotrophic biomass from both reactors are summarized in Table 4.4. The standardized decay coefficients were estimated by adjusting the measured values using the temperature coefficient of ϴ =1.04 according to Metcalf & Eddy (Tchobanoglous et al., 2003). It was found that the decay coefficient (bH) for the ozonated reactor was 0.159 ± 0.025 d-1, which was higher than that for the control (0.132 ± 0.012 d-1). However, t-tests with a specified alpha level of 5% indicated that there was no statistical difference between the decay coefficient for the control reactor and that for the ozonated system. The decay coefficient is a lump parameter for the activated sludge model (ASM No.1). It varies with many factors such as the nature of the wastewater treatment process and the operational conditions. Microbiological endogenous processes are complicated and not fully understood because they cover a group of biological and ecological mechanisms, including endogenous respiration and cell maintenance, cell decay, cryptic growth, predation on bacteria by protozoa, viral attack, and adverse environmental conditions (e.g., pH, toxic substances, and temperature) (Hao et al., 2010). Table 4.5 shows reported decay coefficients under different conditions along with the results of the present study. From Table 4.5, it can be seen that the decay coefficient measured (0.132 d-1) for the control SBR at SRT = 50 d was similar to the results (0.13-1 and 0.14 d-1) previously reported by Zhang and Hall (2006), where the same wastewater for the UBC campus was used as the feed; and it fell within in the range of the decay coefficients proposed by Metcalf and Eddy (Tchobanoglous et al., 2003).    115 Table 4.5  Reported decay coefficients at 20℃ Wastewater Process SRT (d) Decay coefficient Reference Municipal CEBPR (Conventional Enhanced biological phosphorus removal) 17-25 0.14 Zhang and Hall, 2006  Municipal MEBPR (Membrane Enhanced biological phosphorus removal) 17-25 0.13 Zhang and Hall, 2006  Municipal MBR (Membrane bioreactor) 5-30 0.08 Wen et al., 1999 Municipal ASP(Activated sludge process)  0.24 Dold et al., 1986 Municipal ASP  0.4 Kappeler and Gujer, 1992 Municipal Extended aeration Process 20-40 0.019-0.031 Mardani et al., 2011 Pharmaceutical wastewater ASP 10-62.5 0.07 Gupta and Sharma, 1996 Municipal ASP  0.06-0.15 Tchobanoglous et al., 2003 Municipal ASP with ozone treatment 18-30 1.3 Gardoni, et al., 2011 Municipal SBR-MBR 50 0.132 Present Study Municipal SBR-SBR with mixed liquor ozone treatment 50 (nominal) 0.159 Present Study  For the ozone-treated SBR, the decay coefficient measured was 0.159 d-1 which was comparable to the results for conventional activated sludge treatment processes (ASP) reported by some authors (Dold et al. 1986; Kappeler and Gujer, 1992) as noted in Table 4.5. However, the decay coefficient estimated for the ozonated system in the present study was far less than the value of bH = 1.3 d-1 reported by Gardoni et al. (2011), who conducted a full-scale experiment of sludge reduction by means of the ozonation of the sludge recycling stream. Gardoni et al. explained that ozone exposure did not cause acute toxicity, but it did result in sub-lethal damage that increased the average decay rate of the biomass and contributed to 17% sludge reduction. In their study, the ozone was applied in the sludge recycle stream with the dosage varying from 0.8 to 2.5 g O3 per kg of dry solids treated. In the present study, the mixed liquor was ozonated with a dosage of 0.18 – 0.20 g O3/g TSS (0.006 ~ 0.022 g O3 per g TSS treated each cycle) and a lower decay coefficient was observed. It seems that mixed liquor ozonation slightly promoted endogenous 116 processes but it did not cause sub-lethal microorganism damage to significantly affect the endogenous process and the performance of nitrogen and phosphorus removal.   4.3.2 The Maximum Specific Growth Rate and Half-velocity Constant The purpose of sludge reduction is to lower the biomass yield in biological wastewater treatment. The specific growth rate of the heterotrophs is one of the key parameters to estimate sludge production. The specific growth rates of the heterotrophs for the control and the ozonated systems at SRT = 50 d were measured by the batch respirometric methods described in Chapter 2.4.4. The primary effluent from the UBC pilot plant was used as the substrate and 5 mL mixed liquor from each reactor was added as the seed for the batch tests.  The results are shown in Table 4.6. The specific growth rate estimated for the ozonated systems was 4.59 ± 0.55 d-1, which was similar to the specific growth rate for the control system (4.55 ± 0.69 d-1). Table 4.6  The specific growth rates of the heterotrophs for the control and the ozonated  Test # The Control The Ozonated Temperature (℃) µH(d-1) µH(d-1) @20℃ Temperature (℃) µH(d-1) µH(d-1) @20℃ 1 21.8±7 4.34 3.84 21.9±8 5.16 4.54 2 22.1±0.8 4.28 3.71 22.4±0.6 5.12 4.35 3 22.3±0.9 6.03 5.16 22.3±0.9 6.56 5.61 4 24.4±0.7 6.64 4.47 23.3±0.5 6.15 4.57 5 22.8±0.7 6.27 4.87 22.0±0.6 4.97 4.15 6 25.1±0.5 8.44 5.24 24.0±0.1 6.16 4.30 Mean 4.55±0.69 d-1 4.59 ± 0.55 d-1  The maximum specific growth rates and half-velocity constants of the heterotrophic biomass in the control and the ozonated systems at SRT =50 d were measured by the batch respirometric 117 methods described in Chapter 2.4.5. According to Equation 2-9, The variables 1𝜇 and 1𝑆 can be modeled by a linear relationship. The values of the terms  𝐾𝑠𝜇𝑚 and µm can be determined from the slope and the intercept of a plot of 1𝑆 versus  1𝜇. Therefore, the maximum growth rate (µm) and the half velocity coefficient (Ks) for heterotrophic microorganisms can be estimated. Three independent batch tests for each reactor were conducted and the results are shown in Figure 4.6 and Figure 4.7. The measured maximum specific growth rates of the heterotrophs for both reactors are summarized in Table 4.7. The maximum specific growth rate in the ozonated system was 6.21 ± 0.31 d-1, which was statistically lower than the maximum specific growth rate for the control (8.31 ± 0.55 d-1).  Figure 4.6  The plot of 1/S versus 1/µ for estimation of the maximum specific growth rate and half-velocity constant in the control reactor 118  Figure 4.7  The plot of 1/S versus 1/µ for estimation of the maximum specific growth rate and half-velocity constant in the ozonated reactor  Table 4.7  The maximum specific biomass growth rate at 20℃ Test# Control Ozonated µmax(d-1) R2 µmax(d-1) R2 1 8.53 0.991 6.27 0.985 2 8.31 0.983 6.07 0.978 3 8.09 0.953 6.30 0.995 Mean 8.31 ± 0.55 d-1 6.21 ± 0.31 d-1  Table 4.8 shows a selection of maximum specific growth rates of heterotrophs reported in the literature. The µmax of the heterotrophs in the control reactor (8.31 d-1) in the present study was comparable to the results for the MEBPR reported by Zhang and Hall (2006) and the rate proposed by Bruce and Perry (2001). It seems that the maximum specific growth rate of the biomass subjected to mixed liquor ozonation was lower than the average values reported in the literature. This finding is consistent with the results by Gardoni, et al. (2011). It seems that there 119 may be more slow-growing microorganisms in the ozonated reactor, which reduced sludge production. Table 4.8  The reported maximum specific growth rates of the heterotrophs at 20℃ Wastewater Process SRT (d) µmax(d-1) Reference Municipal CEBPR 17-25 11.9 Zhang and Hall, 2006 Municipal MEBPR 17-25 8.36 Zhang and Hall, 2006 Carbohydrate BOD   13.2 Bruce and Perry, 2001 Other BOD   8.4 Bruce and Perry, 2001 Municipal ASP with ozone treatment 18-30 4.5 Gardoni et al., 2011 Municipal SBR 50 8.31 Present Study Municipal SBR with ozone treatment 50 (Nominal) 6.21 Present Study The results of the half-velocity constant measurement are summarized in Table 4.9.  Table 4.9  The Half-velocity constant (mg/L COD) at 20℃ Test # Control Ozonated Ks (mg/L total COD) R2 Ks (mg/L total COD) R2 1 177 0.991 79 0.985 2 152 0.983 111 0.978 3 105 0.953 72 0.995 Mean 145 mg/L total COD 87 mg/L total COD  The average half-velocity constants for the control and the ozonated systems were 145 mg/L total COD (tCOD) and 87 mg/L tCOD, respectively. The fraction of the biodegradable COD that is dissolved and easily biodegradable for municipal wastewater was estimated to be 0.25 (Van Haandel and Van Der Lubbe,  2012). So the average half-velocity constants for the control and for the ozonated were 36 mg/L biodegradable soluble COD (bsCOD) and 22 mg/L bsCOD, 120 respectively. Typical values of half-velocity constants have been reported to be 20 - 60 mg/L bsCOD (Tchobanoglous et al., 2003). The low half-velocity constant for the ozonated system indicates that a low concentration of bsCOD in the effluent could be achieved, which explains why the effluent quality with ozone treatment was not significantly affected in Chapter 3.   4.4 Summary The results of nutrient removal kinetic parameter estimation showed that nitrification and phosphorus removal were not affected significantly in the SBR-MBR system combined with mixed liquor ozonation. The heterotroph kinetic parameters measured in this study were comparable to the results reported by others. There was no statistical difference between the decay coefficient for the control reactor and that for the ozonated system. The maximum specific growth rate of the biomass subjected to mixed liquor ozonation was statistically lower than that of the control, which indicates more slow-growing microorganisms in the ozonated reactor and thus lower sludge yield achieved. The average half-velocity constants for the control and the ozonated systems were comparable. 121 Chapter 5: Active Biomass and Microorganisms Community  The effects on the viable microorganisms and microbial community structure when mixed liquor ozonation is combined with the EBPR system needs to be understood for a full-scale sludge reduction application. In this study, the mixed liquor was sampled, observed and tested to exam the microorganism activity. The molecular tools were used to study the microbial community changes after sludge ozonation.   5.1 Active Biomass To measure the total living biomass quantity in the mixed liquor, cellular adenosine triphosphate (cATP), which represents the amount of ATP contained within living cells was measured. Figure 5.1 shows the total cellular ATP concentrations and the specific cellular ATP concentrations of the mixed liquor in the control reactor and that in the ozonated reactor at SRT = 25 days. The total cellular ATP concentration in the control reactor was 5587 ± 1400 ng/mL, which was significantly higher than that in the ozonated system (3946 ± 1116 ng/mL). The results of t-tests showed that there was a statistically significant difference between the total cellular ATP concentrations for both reactors.  The specific cellular ATP concentrations demonstrate that the mixed liquor in the ozonated reactor hosted a slightly larger fraction of active organisms (2.51 ± 0.33 mg cATP/g VSS) than the control (2.31 ± 0.34 mg cATP/g VSS), although the total cell population was lower due to its lower MLVSS concentration. However, t-tests with a specified apha level of 5% indicated there was no statistical difference between the specific cellular ATP concentrations for both reactors. 122   Figure 5.1  Total and specific cellular ATP for the control and the ozonated systems            at SRT = 25 d Figure 5.2 shows the total and the specific cellular ATP concentrations of the mixed liquor in the two reactors at SRT = 50 days. The total celluar ATP concentration in the control reactor was 7983 ± 1342 ng/mL, which was similar to that in the ozonated (7653±2534 ng/mL) without significant staticstical difference.  The specific cellular ATP concentration of the mixed liquor in the ozonated system was 1.82 ± 0.59 mg cATP/g VSS, which seemed slightly higher than that in the control (1.72 ± 0.28 mg 123 cATP/g VSS). However, t-tests with a specified apha level of 5% indicated there was no statistical difference between the specific cellular ATP concentrations for both reactors at SRT = 50 days.  Figure 5.2  Total and specific cellular ATP for the control and the ozonated systems            at SRT = 50 d Although the specific cellular ATP concentrations at SRT = 50 d (1.72 -1.82 mg cATP/g VSS) were generally lower than those at SRT = 25 d (2.31 – 2.51 mg cATP/g VSS) for both reactors, the biomass activity per unit volume at SRT = 50 d (7653 - 7983 ng/mL) was still higher than 124 those at SRT = 25 d (3946 – 5587 ng/mL) due to about 50% higher MLVSS concentrations at the longer SRT.  5.2 Microscopic Observation 5.2.1 The Flocs Figure 5.3 shows micrographs of sludge flocs from both reactors. It shows that the ozone-treated system exhibited a mixed liquor with essentially no filaments, which was consistent with the obervations reported by Caravelli et al. (2006) and Vergine et al.(2007).   Figure 5.3  Microscopic observation of sludge flocs in two reactors (left: the control, right: the ozone treated) Filamentous microorganisms have large surface areas which give them more exposure to the ozone and its intermediate oxidants and thus, they are more vulnerable to inactivation in the ozonated reactor. The absence of filamentous bacteria made the ozone-treated sludge floc compact and firm, which may account for the significant improvement of sludge settling velocity (Chapter 3).  125 Table 5.1 summarizes the characteristics of flocs from the control reactor and the ozone-treated reactor. The flocs in the control reactor had the following characteristics: irregular shape, open structure, less firm, larger size, and high diversity with few free living cells. The shape of flocs in the ozone-treated reactor was round, and the structure was compact. Due to the mixed liquor ozonation, the size of the flocs was medium, the diversity was comparatively low, and the number of the free living cells was relatively high for the ozone-treated reactor. Table 5.1  The characteristics of flocs observed in sludge of the control and ozonated reactors  Flocs in the control reactor Flocs in the ozonated reactor Shape Irregular Round Structure Open Compact Size Median to Large size(>250 µm) Medium-size (25-250 µm) Diversity High Medium Robust Few free living cells Many free living cells   5.2.2 The Protozoa  One approach for sludge reduction is to exploit protozoa and/or metazoa that prey on the bacteria. The reason is that the energy is lost in the food chain due to inefficient biomass conversion during energy transfer from low to high trophic levels and part of the sludge is converted to liquid or gaseous compounds, and the total biomass production will thus be reduced (Liang et al., 2006; Ratsak et al., 1996). Figure 5.4 and Figure 5.5 show protozoa observed in the mixed liquor in the control reactor and the ozonated reactor, respectively.  126  Figure 5.4  protozoa observed in sludge of the control reactor  Figure 5.5  protozoa observed in sludge of the ozonated reactor 127 Abundant protozoa were observed in the mixed liquor from the control reactor by qualitative assessment. The sludge in the control reactor exhibited a high diversity of protozoa: most common protozoan species in activated sludge processes such as ciliates, flagellates and amoeba were observed (Figure 5.4). However, few protozoa were observed in the sludge from the ozone-treated reactor. This may be due to their exposure to ozone regularly during mixed liquor ozonation. Under this condition, the growth of protozoa was greatly inhibited. Since a large population of protozoa favors sludge reduction, approaches to improve the protozoa population while coupling the mixed liquor ozonation with an EBPR process needs be further studied in the future.  5.3 The Results of the Analysis of Genomic 16S rDNA To understand the effect on community structure when mixed liquor ozonation is combined with the EBPR system, the microorgansims need to be identified and enumerated according to their inheritable genetic content. The most direct approach is to target the 16S rRNA, a powerful phylogenetic marker, which is able to reveal the diversity of the microbial communities.  5.3.1 The Taxonomic Composition  Mixed liquor samples from both reactors were taken and each sample was sequenced twice. All the sequences were compared with the bacterial databases Greengenes (DeSantis et al., 2006) and UniRef50 (Suzek et al., 2015). The control mixed liquor samples yielded about 34,356 tags (the fingerprint of the species) and 460 species were identified; while the ozonated mixed liquor 128 yielded about 40,491 tags and 413 species were identified. The detailed taxonomic results are listed in Appendix B and Appendix C. Figure 5.5 shows the affiliation and distribution of the 16S rDNA of the molecular inventory. The results revealed a different composition of the bacteria community in the control reactor relative to that of the ozonated reactor. The samples of mixed liquor from the control reactor were dominated by the Proteobacteria (including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Delta/espsilonproteobacteria), Bacteroidetes/Chlorobi, and Planctomycetes. Considerable percentages of Chloroflexi, Chlamydiae/Verrucomicrobia, Spirochaetes, Actinobacteria, and Gemmatimonadetes were also found in mixed liquor from the control. The samples from the ozone-treated reactor were dominated by the Proteobacteria (including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Delta/espsilonproteobacteria), Planctomycetes, and Gemmatimonadetes. A significant amount of Chlamydiae/Verrucomicrobia, Fibrobacteres/Acidobacteria, Chloroflexi, Actinobacteria, Bacteroidetes/Chlorobi was also identified in the sludge samples from the ozone-treated reactor. All samples contained about 8.9%~12.7% of uncultured soil bacteria.    129    Figure 5.5  Affiliation and distribution of the 16S rDNA of the molecular inventory  (Up: the control;  Down: the ozonated)   130 5.3.2 The Filaments  About 3.5% of tags were associated with Spirochaetes (Figure 5.5) in the sludge samples from the control reactor, while only few Spirochaetes tags were detected in the samples from the ozone-treated system. The species in the phylum Spirochaetes were axial filaments, and their growth appeared to be inhibited by mixed liquor ozonation, perhaps due to their large surface areas and thus high likelihood of exposure to the ozone. Figure 5.6 shows the taxonomic profile comparison at the family level for samples from the UBC pilot plant (the MBR EBPR process), the influent, the ozone-treated SBR, and the control SBR. The samples from the ozone-treated SBR exhibited a taxonomic profile that differed from the other mixed liquors. The major difference was the Saprospiraceae distribution (the pink color bar in Figure 5.6) in the samples. The Saprosipiraceae is a family of large, filamentous microorganisms identified in activated sludge. While a significant amount of the Saprospiraceae was detected in all the samples from the UBC pilot plant and the control SBR, few Saprospiraceae were identified in the sludge samples from the ozone-treated SBR. The absence of filamentous species in the sludge samples from the ozone-treated reactor was consistent with the microscopic observation mentioned above, which may explain the observed significant improvement of sludge settling. 131  Figure 5.6  The taxonomic profile comparison at Family level  5.3.3 The Nitrifiers The mixed liquor samples from the control SBR contained about 0.32% nitrifier tags, while about 0.38% of nitrifiers tags were identified in the samples from the ozone-treated reactor. Table 5.2 shows the nitrifiers identified in the sludge samples from both reactors. The similar diversity of the nitrifiers in the control reactor and the ozonated system indicates that nitrification was not affected when mixed liquor ozonation was combined with an EBPR system. The species uncultured Nitrosomonadaceae bacterium, uncultured Nitrosospira sp., uncultured Nitrospira sp., uncultured Nitrospirae bacterium, Nitrospira sp., Nitrobacter winogradskyi were identified in the samples from both reactors. The species uncultured Nitrosomonas sp. and Nitrosomonas sp. NM 41 were only detected in the samples from the control reactor, while the species Nitrospira defluvii, Nitrosomonas sp., and Nm59 uncultured Nitrospiraceae bacterium were identified in the samples from the ozone-treated one. 132 Table 5.2  The nitrifiers identified in sludge samples from both reactors Nitrifiers identified in the control reactor Nitrifiers identified in the ozone-treated reactor uncultured Nitrosomonadaceae bacterium  uncultured Nitrosomonadaceae bacterium  uncultured Nitrosospira sp. uncultured Nitrosospira sp. uncultured Nitrosomonas sp. Nitrospira defluvii  uncultured Nitrospirae bacterium  uncultured Nitrospirae bacterium uncultured Nitrospira sp. uncultured Nitrospira sp. Nitrobacter winogradskyi Nitrobacter winogradskyi uncultured Nitrobacter sp. uncultured Nitrospiraceae bacterium Nitrosomonas sp. NM 41 Nitrosomonas sp. Nm59 Nitrospira sp. Nitrospira sp.  5.3.4 The PAOs The major PAOs Candidatus Accumulibacter sp. (Hesselmann et al., 1999) and Candidatus Halomonas phosphatis (Nguyen et al., 2012) were identified in the mixed liquor samples from both reactors. It was found that a higher percentage of the species Candidatus Accumulibacter sp. occurred in the samples from the control reactor (2.12%) than in the samples from the ozone-treated one (0.83%); while a significantly higher number of the species Candidatus Halomonas phosphatis was found in the samples (69 tags) from the ozone-treated reactor than that (7 tags) from the control. This may explain why phosphorus removal efficiency was not affected by mixed liquor ozonation.   5.3.5 Microbial Diversity  Microbial diversity or alpha diversity, which was measured by using Shannon’s diversity index, is shown in Figure 5.7. The Shannon diversity index is an index that is used to characterize species diversity in a community. As a strong chemical oxidant, ozone is able to damage and 133 inactivate microorganisms in mixed liquor, particularly those species with high surface areas, so the growth of these species may be inhibited due to their high chance of exposure to the ozone treatment. Therefore, the sludge samples from the ozone-treated SBR exhibited lower diversity than those from the control reactor and from the UBC pilot plant.   Figure 5.7  Shannon diversity Index   5.4 Summary The results of the cellular ATP tests show that specific cellular ATP concentration in the ozonated reactor was at the same level as that in the control, although the total active biomass in the ozonated reactor was lower, which indicates that microorganism activity was not significantly affected when mixed liquor ozonation was applied in the EBPR system. 134 Essentially no filaments and a low microbial diversity were found in mixed liquor from the ozonated reactor by the microscopic observation, which was further confirmed by the molecular tools. The results of the analysis of genomic 16S rDNA revealed a different bacterial community in the control reactor and the ozonated system. The same PAOs and nitrifiers were identified in both reactors which supported the conclusion that the phosphorus removal and nitrification were not affected by mixed liquor ozonation in the EBPR system.   135 Chapter 6: The Mixed Liquor Ozonation Test  6.1 Introduction 6.1.1  Sludge disintegration by ozone treatment with a high degree of suspended solids solubilization at a low dosage is desirable. However, sludge ozonation is a complicated process and many factors affect its efficiency. In the present study, a plug-flow ozone reactor was used to test the performance of sludge ozonation with different TSS concentrations, three types of sludge, with or without carbonate or bicarbonate addition, and under high and low pH conditions.  As a strong chemical oxidant, ozone can be used to disintegrate excess sludge and a high degree of particulate solubilization can be achieved (Muller, 2000). The main drawback for sludge ozonation is its high energy consumption and associated high operating cost. The ozone dosage, generally expressed by g O3/g TSStreated, is one of the most important parameters to evaluate the efficiency of sludge ozonation. A low dosage with a high degree of disintegration is desirable. However, the dosages reported in the literature vary greatly. For example, it was reported that an ozone dosage lower than 0.015 g O3/g VSS was not sufficient to cause cell rupture (Albuquerque et al., 2008); no alteration of bacterial DNA was detected at the dosage of 0.02 g O3/g TSS and destruction of bacteria occurred mainly at an ozone dosage above 0.08 g O3/g TSS (Yan et al., 2009). By measuring oxygen uptake rate (OUR), at an ozone dosage of around 0.02 g O3/g TSS, 80% of microbial respiration activity was observed to be lost (Chu et al., 2008). Another study (Saktaywin et al., 2005) reported that around 70% of sludge bacterial activity was lost at an ozone dosage of 0.03-0.04 g O3/g TSS.  136 The discrepancies reported in the ozone dosages required for sludge disintegration may be due to many other factors which may be overlooked. Sludge is a three-phase, complex matrix of flocs, living cells and organo-mineral materials, and the interactions between ozone and activated sludge are complicated and poorly understood (Dziurla et al., 2005). In addition to the ozone dosage, the efficiency of sludge ozonation may depend on many other factors including (1) sludge characteristics, (2) ozone mass transfer efficiency in the reactor and the configuration of the ozone reactor, (3) flow rate and concentration of ozone in the gas-phase, (4) flow rate and suspended solids concentration in the treated sludge, (5) contact time between ozone and sludge, (6) scavengers in sludge, and (7) the pH. In the present study, three different types of sludge were tested to investigate the sludge disintegration efficiency. Three different suspended solids concentrations of mixed liquors were also tested with the same amount of ozone applied. The batch test operating conditions were described in Chapter 2. The effects of the carbonate and bicarbonate (as O3 scavengers) addition and pH adjustment on the performance of sludge ozonation were also tested. Hydroxyl radicals (OH°) generation during ozonation was assumed to be one of the critical factors for sludge solubilization and cellular inactivation (Dziurla et al., 2005; Yan et al., 2009). Carbonate and bicarbonate ions are the most common scavengers in mixed liquor. Carbonate and bicarbonate react with OH°, stopping the production of superoxide radicals (O2°¯/HO2°), and terminating the chain reaction. At high pH conditions, the main reaction in sludge ozonation is the hydroxyl radical reaction; and at low pH conditions, the ozone reaction is dominated by direct ozone oxidation reactions. In the present study, batch tests were conducted to investigate sludge disintegration with the addition of carbonate/bicarbonate, base and acid. 137  6.2 The Effect of Solids Concentration  The efficiency of sludge ozonation was related to the suspended solids concentration. Figure 6.1 shows the efficiencies of sludge disintegration from the UBC pilot-scale MEBPR process by ozonation at three different suspended solids concentrations. It seems that more extensive sludge solubilisation and cellular inactivation were achieved with the lower suspended solids concentrations.  Since the same amount of ozone was applied to each sludge sample, the ozone dosage, if defined as the mass of ozone applied per mass of treated TSS, increased from 5.4 to 10.9 mg O3/g TSS when TSS concentration was decreased from 11,580 mg/L to 5699 in Figure 6.1(A). However, the highest soluble COD was not achieved at the lowest TSS concentration. The highest concentration of soluble COD after ozone treatment was measured with TSS = 8740 mg/L. It seemed that there existed an optimum TSS concentration at which the maximum soluble COD could be achieved.  The extents of TSS and VSS reduction/solubilization generally increased when the suspended solids concentration of the treated sludge was decreased, which is illustrated in Figure 6.1 (B) and (C). However, the percentage of suspended solids solubilized did not significantly increase even when the concentration of suspended solids was lower (the ozone dosage increased).  Figure 6.1(D) shows that sludge from the pilot-scale MEBPR process was highly susceptible to ozone treatment and about 44.9% of the living cells in sludge were inactivated (which was measured by the difference of the cellular ATP concentrations before and after treatment) even at 138 the lowest dosage of 5.4 mg O3/g TSStreated. The cellular inactivation fraction increased when the TSS concentration was decreased.  In the present study, the ozone dosage was defined by the the mass of ozone applied per mass of treated TSS, which is the same approach as that generally reported in the literature. It could be more accurate to use the mass of ozone actually consumed during sludge ozone treatment to estimate the ozone dosage. Further research with well controlled experiments is needed to further verify the results of the present study.  139  Figure 6.1  Sludge disintegration by ozone treatment at three different suspended solids concentrations 140 6.3 The Effect of Sludge Source The source of the mixed liquor had a significant impact on sludge solubilisation and cellular inactivation during batch sludge ozonation. Figure 6.2 shows the impact of sludge ozonation for three different types of sludge at different ozone dosages. At each ozone dosage, the percentage of cells inactivated was greater than the percentage of particulates solubilized. In addition, sludge from the SBR process exhibited a lower degree of solubilisation and cellular inactivation than did sludge from the MEBPR process, even at a lower TSS concentration and a high ozone dosage. Sludge from the SBR that was combined with sludge ozonation, exhibited the lowest degree of sludge solubilisation and cellular inactivation, even though the TSS was the lowest tested and therefore, the ozone dosage was highest. The particle size distribution of the mixed liquors may also have been a factor affecting the performance of sludge ozonation. Figure 6.3 shows the particle size distribution of three mixed liquors expressed by volume, respectively. The median diameters of the particles in sludge from the MEBPR, the control SBR and the ozonated SBR were 23.0 µm, 128.1 µm and 87.0 µm, respectively. The particle size of the MEBPR sludge was significantly smaller than that of sludge from SBRs, and therefore higher degrees of sludge solubilisation and cellular inactivation were observed.  141  Figure 6.2  Sludge solubilization and cellular inactivation vs. ozone dosage for different TSS concentrations and different types of sludge The ozone mass transfer mechanism in gas/liquid systems is generally explained by the “fast kinetic regime” theory proposed by Danckwerts (1970). The rate limitation occurs in the liquid film, where the ozone can react quickly with the various dissolved organic substances and thus be consumed. This causes the apparent rate of ozone mass transfer to exceed the maximum rate of physical gas-liquid mass transfer. Normally, the thickness of the liquid film is between 15 to 20 μm. During sludge ozonation, with the enhancement by the fast reaction, the effective thickness of the film would be reduced to less than 10 μm (Paul and Debellefontaine, 2007). The thin liquid film allows only smaller particles and/or the peripheries of large flocs to be oxidized by ozone, while the cells inside the flocs may remain viable due to the physical protection 142 provided by the aggregate structure (Chu et al., 2009). The theory may explain why the efficiency of ozonation of the MEBPR sludge was higher than that for sludge from the SBRs. However, it cannot explain why a lower TSS/VSS solubilization percentage was achieved for sludge from the ozonated SBR than from the control SBR, even though the ozonated SBR supported a smaller particle size distribution than that of the control (87.0 µm versus 128.1 µm).  Figure 6.3  The particle size distribution by volume for three types of sludge  The photos of three types of sludge from microscopic observation (Figure 6.4) may explain the discrepancy. Figure 6.4 illustrates the structural differences among the three types of sludge. The MEBPR sludge was composed of many small loose flocs, fine particles and filamentous bacteria (Figure 6.4(A)). Flocs of large sizes were observed in sludge from both SBRs (Figure 6.4 (B) and (C)). Filamentous bacteria were obvious in sludge from the MEBPR and the control SBR. No filamentous bacteria were observed and the flocs were compact in sludge from the ozonated 143 SBR, which was similar to the observations reported by other researchers of  sludge ozonation (Nilsson et al., 2014). The loose structure of flocs and the larger surface areas provided by filamentous microorganisms may enhance the contact with ozone and/or hydroxyl radicals; and thus a greater extent of sludge disintegration and cellular inactivation could be achieved. The microorganisms from the ozonated SBR may be adapted to long-term ozone exposure and as a consequence, they formed solid and compact flocs which may have been able to resist the attack from ozone. Therefore, the lowest degree of TSS and VSS solubilization and the lowest extent of cellular inactivation were achieved for sludge from the ozonated SBR.  Figure 6.4  The microscopic photos of three types of sludge   144 6.4 The Effects of Carbonates/Bicarbonates and pH on Sludge Ozonation 6.4.1 The Effects of Carbonates/Bicarbonates The results of sludge disintegration experiments with mixed liquor from the control SBR, with the addition of carbonates, sodium hydroxide and hydrochloric acid and with and without ozone are shown in Figure 6.5. Figure 6.6 shows the results of similar sludge disintegration experiments with mixed liquor from the SBR combined with the ozone treatment..  Figure 6.5  The sludge solubilization and cellular inactivation of sludge from the control SBR process with carbonate/bicarbonate, base, and acid addition with/without ozone The carbonates/bicarbonates addition resulted in impacts on sludge ozonation. In Figure 6.5, high cell inactivation was observed with sodium carbonate addition, which may have been due to a pH increase. About 6.9% of the TSS and 5.5% of the VSS were solubilized. The cellular inactivation fraction was 67.8%, which was significantly higher than that with sludge ozone treatment only. When bicarbonate was added to the sludge prior to ozonation, the proportion of  TSS solubilized was reduced from 11.4% to 9.8% and the VSS solubilization decreased from 145 11.2% to 9.9% compared to those achieved with ozone treatment only. The carbonates/bicarbonates normally act as hydroxyl radical scavengers which reduce ozone radical reactions, which, in turn, was expected to reduce the efficiency of sludge ozonation. However, the cellular inactivation efficiency increased from 22.6% to 73.4%, a result that may have been associated with the increased pH (10.4) due to carbonates addition. It seems that a high concentration of carbonates/bicarbonates may have a dual effect on the performance of sludge ozonation: they may not only act as hydroxyl radical scavengers to reduce ozone indirect reaction but also as promotion initiators (OH-) due to the associated pH increase.   Figure 6.6  The sludge solubilization and cellular inactivation of the sludge from the SBR combined with the ozone treatment process with added carbonate/bicarbonate, base, and acid with/without ozone  6.4.2 The Effects of pH Either alkali or acid addition alone can disintegrate sludge. In the present series of batch tests, a high degree of sludge solubilization was achieved by mixing sodium hydroxide or hydrochloric 146 acid with the mixed liquors from both systems. For TSS, the observed reduction was 13.8 - 23.7% at high pH and 12.3% - 16.1% at low pH. These ranges were higher than those achieved by ozonation alone (8.4% - 11.4%). For VSS, reductions of 13.8 - 16.1% at high pH and 7.1 - 14.6% at low pH were observed, which was also comparable to or slightly higher than those with ozonation only (7.4 - 11.2%). Low pH and high pH treatment tended to inactivate a high percentage of cells. For sludge from the control SBR (Figure 6.5), about 22.6% of the cells were inactivated by ozone treatment only, while 67.8%, 61.5%, and 87.1% of cells were inactivated by carbonates, sodium hydroxide and hydrochloric acid treatment, respectively. For sludge from the ozonated SBR (Figure 6.6), additional ozone treatment only inactivated 19.2% of cells, while bicarbonates, sodium hydroxide and hydrochloric acid treatment inactivated 34.5%, 52.7%, and 97.6% of cells, respectively. Among all sludge disintegration methods tested, sludge ozonation alone achieved the lowest cellular inactivation efficiency. Although sludge flocs tended to be resistant to ozone treatment, the sludge was still susceptible to low and high pH conditions. In addition, the cells in sludge might be more tolerant of high pH (~10) than low pH (~2) treatment since a lower cellular inactivation efficiency was observed under high pH conditions than under low pH conditions.  When sludge ozonation was combined with high pH treatment, the degree of TSS and VSS solubilization was significantly improved. The high pH favors hydroxyl radical generation. Because the hydroxyl radical is one of strongest oxidants and the sludge ozonation mechanism may be dominated by radical chain reactions, the efficiency of sludge disintegration might be improved. In Figure 6.5 and Figure 6.6, sodium hydroxide addition combined with ozone treatment achieved the highest degrees of TSS and VSS solubilization among all methods used. Even if bicarbonates acted as radical scavengers during sludge ozonation, the high pH they 147 caused might improve the TSS and VSS solubilization (14.0% versus 8.4% for TSS and 11.1% versus 7.4% in Figure 6.6). When sludge ozonation was combined with acid addition, the efficiencies of TSS and VSS solubilization increased. However, the values were lower than those achieved under high pH conditions. For cellular inactivation, more than 90% of cells were inactivated in both cases. These were the highest efficiencies observed among all the sludge disintegration experiments completed. It is possible that low pH may be more lethal to microorganisms than high pH conditions.   6.4.3 Soluble COD Release A summary of measurements of  the soluble COD released during sludge disintegration treatment are shown in Figure 6.7. The soluble COD released by ozonation alone was low compared to those observed with high pH and low pH treatments. The small amount of soluble COD released may be due to the low cellular inactivation percentage achieved (19.2% for sludge from the control SBR and 22.6% for sludge from the ozonated SBR in Figure 6.5 and Figure 6.6) after ozone-only treatment. The low cellular inactivation efficiency indicates that few cells were disrupted by ozonation and therefore, the quantity of organic material released was low.  Figure 6.7 also indicates that the highest released soluble COD concentration was achieved by the combination of ozonation and high pH treatment. As discussed above, the high pH favors the hydroxyl radical chain reaction, so that the efficiency of sludge disintegration and the release of COD were significantly improved.   148  Figure 6.7  Soluble COD release by ozonation, adding carbonate/bicarbonate, base, and acid with and without ozone  The soluble COD concentration was lower for sludge treated by ozonation combined with hydrochloric acid treatment than that with hydrochloric acid treatment only. However, in Figure 6.5 and Figure 6.6, the TSS and VSS solubilization percentages observed were higher with ozonation combined with acid treatment, than with acid treatment only. It is possible that ozone mineralized some soluble organic matters which were released by direct reaction under low pH conditions, and a lower concentration of soluble COD was observed. The released soluble COD concentrations were generally comparable for sludge from the control SBR and those from the ozonated SBR (Figure 6.7). The soluble COD concentration was higher with carbonates treatment than that by bicarbonates addition, which may be due to carbonates’ higher pH and less effectiveness as radical scavengers than bicarbonates.   149 6.4.4 The Effects on Particle Size Distribution Figure 6.8 shows the particle size distribution by volume before and after sludge disintegration treatment. The peaks of the curves slightly shifted to left (indicating smaller particles) after treatment, which indicates that flocs were disrupted and smaller particles were generated. The curve for ozonation combined with NaOH treatment exhibited the biggest shift, which was consistent with the highest TSS and VSS solubilization efficiencies achieved with this treatment.  Figure 6.8  Particle size distribution by volume  Figure 6.9  Particle size distribution by number 150 Figure 6.9 shows particle size distribution by number for different treatment methods. The particle size distribution by number after treatment of sludge from the control SBR exhibited significant changes. It is possible that the large-size flocs in sludge from the control SBR were disintegrated to many small flocs and the small-size free bacteria were solubilized, which made the peak of the curve lower (more uniform) and shifted to right side (less free bacteria and more small flocs). For sludge from the ozonated SBR, the flocs were compact. It’s difficult to disintegrate the flocs and the particle size distribution by number was not significantly changed by treatment.  6.5 Summary The efficiency of sludge ozonation depends on many factors, including TSS concentration, sludge type, the presence of carbonates and bicarbonates, and the system pH. Mixed liquor ozonation with a low TSS concentration might achieve a high degree of sludge solubilisation and high cellular inactivation efficiencies. There may be an optimum TSS concentration at which the maximum soluble COD can be achieved. The source of sludge exhibited a significant impact on sludge solubilisation and cellular inactivation by sludge ozonation. Both the particle size distribution and the structure of flocs had significant impacts on the sludge disintegration efficiencies achieved.  Carbonate or bicarbonate addition during ozone treatment may reduce the efficiency of sludge disintegration. The degree of solids solubilization was significantly improved under high pH conditions. Under low pH conditions, mixed liquor ozonation may achieve more than 90% 151 cellular inactivation. The cells in mixed liquor from the ozonated SBR may be more resistant to treatment at high pH than at low pH.   152 Chapter 7: The Zero Sludge EBPR System with Mixed Liquor Ozonation  Although zero excess sludge wasting has been achieved in a conventional secondary activated sludge process when it was combined with sludge ozone treatment, no zero sludge EBPR processes have been reported due to the problem of phosphorus accumulation in the system. In the present study, a zero sludge EBPR process at lab-scale was explored by combining a mixed liquor ozonation unit and a phosphorus removal and extraction process with the EBPR system.  7.1 The Zero Sludge EBPR System Coupled with Mixed Liquor Ozonation The SBR MBR system with a mixed liquor ozone-treatment unit described in Chapter 2 was operated without sludge wasting for 136 days. The experiment started on June 21st and ended on Nov 3rd, 2016. The filling ratio was 1/3 and the HRT was 16 hours. About 7.5 L/cycle or 30 L/d of wastewater was treated by the system. The suspended solids discharged with the effluent during the decanting stage of the SBR were retained in the downstream membrane filtration tank, and returned to the SBR every week. A 10 mL sample of mixed liquor was taken from the SBR during the aerobic stage for TSS and VSS analysis twice a week. The mass of suspended solids removed by sampling amounted to only 1/2250 of the total inventory of suspended solids in the system.  Although zero sludge wasting has been reported from a study with a conventional secondary treatment process coupled with a return sludge ozone-treatment process in a previous study (Lee 153 et al., 2005; Sakai et al., 1997; Yasui et al., 1996), it was expected that this approach could be problematic with an EBPR process. If sludge is not wasted from an EBPR system it was thought that phosphorus might accumulate to excessive levels in the treatment system or break through to the effluent. Further, it was thought that the PAOs may lose their activity if the nominal SRT is infinite. Therefore, it was assumed that there must be an extraction of phosphorus accumulated in the system to maintain the phosphorus mass balance in a zero sludge EBPR system.  A possible solution for the zero sludge SBR EBPR system is illustrated in Figure 7.1. The right hand panel of the diagram illustrates the SBR process coupled with an ozone-treatment unit; and the left panel shows the phosphorus release and discharge process. The phosphorus release and discharge process was operated once every 1 to 2 weeks of SBR operation when a significant amount (0.7 ~ 1.5 g in a 20 L reactor) of phosphorus had accumulated in the system. To release the phosphorus stored in the cells of the PAOs, a VFA solution and municipal wastewater were added to the SBR to the full-tank water level after decanting stage in the SBR. The contents were completely mixed for 2 hours under anaerobic conditions. This was followed by about 1 hour of settling to produce a supernatant containing a high concentration of ortho-phosphate that was then pumped out of the system. The supernatant may be further treated or the phosphorus in the supernatant may be recovered by current commercially available P recovery techniques. The effluent stored in the effluent tank was pumped back to the SBR to maintain the lower water level and to keep the same P loading from the wastewater in the SBR to start a new cycle if more supernatant was pumped.  A simple mass balance calculation for the phosphorus in the system can be calculated as follows. Assuming the average ortho-phosphorus concentration in the influent (wastewater from the UBC community) is 5 mg/L and the average concentration in the treated effluent is 0.1 mg/L, about 154 147 mg/d or 1029 mg/week of phosphorus will be accumulated in the system when no sludge is wasted from the system. According to the results of the earlier P release batch tests (Chapter 4), up to 80 mg/L ortho-phosphate can be achieved under anaerobic conditions by adding VFA and reacting for 2 hours. With an ortho-phosphate concentration of 70 mg/L produced in this manner, about 14.7 L supernatant discharge per week would avoid phosphorus accumulation in the system. 155  Figure 7.1  The diagram of P release and discharge in a zero sludge SBR System  156 7.2 The Ozone Dosage and the Suspended Solids Inventory To achieve zero excess sludge production, the suspended solids inventory should be dynamically balanced with proper ozone dosage. Figure 7.2 shows the ozone dosage and the concentrations of TSS and VSS in the system. The ozone dosage was applied during a 1 minute ozone-treatment per cycle (about 321 mg O3/day) from June 21st to August 7th. During this period, the suspended solids inventory increased continuously without sludge wasting. The ozone-treatment time was increased from 1 minute to 1.5 minutes per cycle (about 482 mg O3/day) from August 8th to September 7th after which the TSS and VSS inventories decreased, indicating that with ozonation for 1.5 minutes per cycle, the system was overdosed. On September 8th, the ozone dosage was reduced to 1 minute per cycle and the TSS and VSS inventories increased accordingly. In October, the ozone treatment time was set to 70 s per cycle (about 375 mg O3/day). At end of this experiment (136 days), the ozone treatment time was set to 30 s per cycle, and the suspended solids significantly increased. The maximum concentrations of TSS and VSS achieved were 9170 mg/L and 7600 mg/L respectively.  Both the suspended solids levels in the influent and the ozone dosage affected the solids inventory during operation with zero sludge wasting. To maintain a stable concentration of suspended solids in the system, the ozone dosage is expected to dynamically change with wastewater characteristics. However, it is a challenge to adjust the ozone dosage manually. From the results of this experiment shown in Figure 7.2, it seems that 70 s per cycle (375 mg O3/day) was near the optimum ozone dosage to maintain a relatively stable inventory of suspended solids without sludge wasting in the system.  Figure 7.3 shows the VSS and TSS concentrations and the VSS/TSS ratios. The VSS/TSS ratios were generally stable with an average value of 0.8. The VSS/TSS ratio did not change with the 157 inventory changes of suspended solids. When ozone dosage was less than 90 s per cycle, the VSS/TSS ratio was not affected. The stable VSS/TSS ratio indicated that inorganic material did not accumulate in the system when a proper dosage ozone was applied to the mixed liquor.  Figure 7.2  The ozone dosage and the concentrations of TSS and VSS in the system   Figure 7.3  TSS and VSS concentrations and VSS/TSS ratio  158 7.3 The Effluent Quality 7.3.1 COD Figure 7.4 shows the measured COD concentrations in samples collected during the zero sludge wasting experiment. The effluent (permeate) concentrations of COD were less than 50 mg/L and about 90% total COD removal was achieved consistently. This demonstrates that a high COD removal efficiency can be achieved in a zero sludge EBPR system operated with mixed liquor ozonation.  Figure 7.4  Total COD of the influent and the effluent  7.3.2 Inorganic Nitrogen Figure 7.5 shows the ammonium-N and nitrate/nitrite-N (NOx-N) concentrations in the influent and the effluent during the experiment. The effluent concentrations of ammonium were low and removal efficiency was greater than 95%, which indicates that nitrification was not affected in the system.  159  Figure 7.5  Inorganic nitrogen concentrations of the influent and the effluent  7.3.3 The Ortho-Phosphate Figure 7.6 shows the measured ortho-phosphate concentrations in the influent wastewater and in the effluent. Biological phosphorus removal was not achieved at the beginning of the experiment. The possible reason was that the EBPR seed sludge used to start the system had been stored in the fridge for about one month before use. Most of PAOs may have lost their activity. Since the seed EBPR sludge contained a high concentration of phosphorus, a significant amount of phosphorus was released from the sludge to the liquid, which caused high ortho-phosphate concentrations in the effluent during the first month of the experiment. In August, unstable P removal was observed that may have been due to the overdosed ozone treatment (from 60 s per cycle to 90 s per cycle) which affected the growth of PAOs. The phosphorus removal efficiency improved after 80 days (early September) and PAOs recovered their activity. Low ortho-010203040500.0010.0020.0030.0040.0050.009/7/2016 8/8/2016 7/9/2016 7/10/2016 6/11/2016NOx-N (mg/L) NH4-N (mg/L) The Influent (NH4-N) The Effluent (NH4-N) The Effluent (NOx)160 phosphate concentrations in the effluent were measured thereafter and more than 85% P removal was achieved.  Figure 7.6  The ortho-P concentrations of the influent and the effluent  7.4 The Batch Operation of Phosphorus Release and Discharge from the System  To avoid phosphorus accumulating in the system, a series of batch experiments for phosphorus release and discharge was conducted at a 1 to 2 week frequency beginning in September, when biological phosphorus removal had been achieved. The procedure was illustrated above in Figure 7.1. The phosphorus removal process started after the idle stage of the SBR cycle. About 1 liter of 14 g/L acetate solution and 5 liters of wastewater were added to the SBR to return the liquid volume to the normal water level. Then the reactor was completely mixed without aeration for 2 161 hours. The phosphorus released under the anaerobic conditions was then removed from the system by supernatant discharge after 1 hour of settling. Table 7.1 shows the results of 6 batch experiments for phosphorus release and discharge. Four phosphorus release batch experiments were conducted after 7 days of normal SBR operation without sludge wasting. About 577 ~ 715 mg of phosphorus were removed with the supernatant discharge from the system each time. The concentrations of the ortho-phosphate in the supernatant were 36 ~ 44 mg/L.  A fifth batch P-release experiment was then conducted after 10 days of normal SBR operation. As indicated in Table 7.1, 996 mg of phosphorus were removed from the system with 15 L supernatant discharge. The last P-release test was completed after 17 days of SBR operation without sludge wasting. In this case, about 1580 mg phosphorus was removed from the system with 17 L supernatant discharge. In this final experiment, the ortho-phosphate concentration in the supernatant was 98.8 mg/L, which would have been suitable for further phosphorus recovery (Adnan, 2003). Table 7.1  Results of batch tests for phosphorus release from zero EBPR system  Date P release Interval (days) Volume of Supernatant (L) Ortho-P concentration (mg/L) Ortho-P released (mg) The remained Ortho-P in the reactor after each batch P-release (mg) 2016-09-22 7 14.5 44.0 645 111 2016-09-29 7 20.0 42.9 622 114 2016-10-06 7 20.0 35.8 715 16 2016-10-16 10 15.0 66.4 996 -39 2016-11-02 17 16.0 98.8 1581 -100  162 Figure 7.7 shows the cumulative phosphorus loading in the influent and the cumulative discharges of phosphorus in the treated effluent and in the supernatant discharged by each batch P-release experiment. From the Figure 7.7, it can be seen that the cumulative P loading in the influent was roughly equal to the cumulative P in the effluent plus the cumulative P removed by the batch P-release and discharge experiments.  Figure 7.8 shows the relative ortho-P inventory in the system during the test. It confirmed that the ortho-P inventory can be maintained and the accumulated P can be controlled by batch P-release and discharge operations. The phosphorus release and discharge by batch operation with VFA addition can maintain a P mass balance in a zero sludge EBPR system combined with a mixed liquor ozone-treatment unit.  Figure 7.7  The cumulative P and P-release 163  Figure 7.8  The relative P inventory in the system  7.5 Summary In this chapter, a zero sludge EBPR SBR process at lab-scale was tested with an ozone-treatment unit and with phosphorus release and discharge once every 1 to 2 weeks. The optimum ozone dosage was observed to be about 70 s per cycle (375 mg O3/day) to maintain a dynamic balance of suspended solids in the system without sludge wasting.  The VSS/TSS ratio was not affected when ozone dosage was less than 90 s per cycle. COD removal, nitrification and phosphorus removal efficiencies were not affected during the experiment. 164 By applying the batch P-release and discharge operations with VFA addition once every 1 to 2 weeks, the problem of P accumulation in the reactor could be avoided and the P mass balance could be maintained in a zero sludge EBPR system. 165 Chapter 8:   The Conclusions  8.1 Introduction The conventional activated sludge process generates a large amount of excess sludge, resulting in significant costs of treatment and disposal and environmental degradation. Excess sludge reduction or minimization in wastewater treatment is one of most challenging tasks in the environmental engineering field. In this comparative study, a combination of mixed liquor ozone treatment and an EBPR process with long SRT operation (SRT = 25 d and SRT = 50 d) was tested to reduce sludge yield. The effects on the effluent quality, microbial activity and community structure, and kinetic parameter values were studied. A new zero sludge EBPR process with a phosphorus release and extraction process was proposed and tested.  8.2 Overall Conclusions • Sludge Reduction It was observed that mixed liquor ozone treatment applied to an EBPR process may be a feasible way to reduce sludge production. At SRT = 25 d, the observed sludge yield of the ozonated reactor was 0.177 g TSS/ g CODremoved (0.145 g VSS/ g CODremoved), which was 35% lower than that in the control reactor (0.273 g TSS/ g CODremoved (0.224 g VSS/ g CODremoved)). At SRT = 50 d, the observed sludge yields of the control reactor and the ozonated one were 0.081 g TSS/ g CODremoved (0.064 g VSS/ g CODremoved) and 0.047 g TSS/ g CODremoved. (0.036 g VSS/ g 166 CODremoved), respectively. A 42% reduction was achieved. Overall an equivalent to about 80% sludge reduction was achieved compared with the control system operated at SRT = 25 d.   • Effluent Quality It was observed that both the control and the ozone-treated systems at SRT = 50 d achieved about 90 ± 1% COD removal, 99 ± 1 % ammonia removal, 79 ± 1% inorganic nitrogen removal, and about 94 ± 1% ortho-P removal. The effluent quality from the ozone-treated system was comparable to that from the control at the long SRT, which indicated that low-dosage mixed liquor ozone treatment applied to an EBPR process may not affect the effluent quality.  • Sludge Properties The sludge settling properties with ozonation were significantly improved compared with the control. The low SVI and high ZSV of mixed liquor from the ozonated reactor indicated improved sludge settling properties at both SRT = 25 d and SRT = 50 d. The improved sludge settling with mixed liquor ozonation suggests that high mixed liquor suspended solids concentrations could be maintained in conventional activated sludge system with secondary sedimentation.  • Nitrification The specific nitrification rate of mixed liquor in the ozonated system was similar to that of sludge in the control. At SRT = 25 d, the ammonium removal rates were 4.21 - 4.71 g NH4+-N /g 167 VSS∙hr for the control reactor and 5.41 - 6.73 g NH4+-N /g VSS∙hr for the ozonated system. At SRT = 50 d, the ammonium removal rate was estimated to be 2.77 - 6.02 g NH4+-N /g VSS∙hr for the control reactor and 4.03 - 6.06 g NH4+-N /g VSS∙hr with ozonation. The volumetric nitrification rate of the mixed liquor from the ozonated reactor was slightly lower than that of the control system due to a lower MLVSS concentration and thus a smaller population of nitrifiers.   • Phosphorus Removal The phosphorus removal rate was not affected in the ozonated system. The maximum P-release rate from the ozonated reactor (13.68 - 16.22 mg P/g VSS∙h) was significantly higher than that of the control (7.15 - 8.05 mg P/g VSS∙h) at SRT = 25 d. At SRT = 50 d, the specific P-release rate in the ozonated reactor (12.37 - 18.64 mg P/g VSS∙h) was similar to that observed in the control (8.39 - 16.68 mg P/g VSS∙h). The specific P-uptake rate (12.57 - 13.40 mg P/g VSS∙h) in the ozonated reactor was more than two times that in the control (3.93 - 4.66 mg P/g VSS∙h) at SRT = 25 d. At SRT = 50 d, the specific P-uptake rate of the ozonated reactor (3.32 - 6.19 mg P/g VSS∙h) was comparable with that of the control (3.21 - 7.07 mg P/g VSS∙h).   • Heterotroph Kinetics  There was no statistical difference between the decay coefficient for the control reactor and that for the ozonated system. The specific growth rate with ozonation was 4.59 ± 0.55 d-1, which was similar to the specific growth rate for the control (4.55 ± 0.69 d-1). 168 The maximum specific growth rate for the ozonated system was 6.21 ± 0.31 d-1, which was less than the maximum specific growth rate for the control (8.31 ± 0.55 d-1). The average half-velocity constant for the control system was 145 mg/L tCOD (36 mg/L bsCOD) and that for the ozonated reactor was 87 mg/L tCOD (22 mg/L bsCOD) at SRT = 50 d, respectively if 0.25 is assumed to represented the fraction of easily biodegradable COD.   • Biomass Activity It was demonstrated that the mixed liquor in the ozonated reactor hosted a slightly larger fraction of active organisms than the control, although the total biomass in the ozonated reactor was lower. At SRT = 25 d, the total biomass in the control reactor (5587 ± 1400 ng cATP/mL) was higher than that in the ozonated system (3946 ± 1116 ng cATP/mL). However, the mixed liquor in the ozonated reactor hosted a similar fraction of active organisms (2.51 ± 0.33 mg cATP/g VSS) relative to that of the control (2.31 ± 0.34 mg cATP/g VSS). At SRT =50 d, the total biomass in the control reactor (7983±1342 ng cATP/mL) was similar to that in the ozonated (7653±2534 ng cATP/mL) and the specific cellular ATP concentration of the mixed liquor in the control (1.72 ± 0.28 mg cATP/g VSS) and that in the ozonated one (1.82 ± 0.59 mg cATP/g VSS) were at the same level without staticstical difference.   • Microbial Community The results of the analysis of genomic 16S rDNA revealed a different composition of the bacterial community in the control reactor than in the ozonated one. The mixed liquor from the ozonated reactor hosted higher percentages of Alphaproteobacteria, Planctomycetes, 169 Gemmatimonadetes than that from the control; while the mixed liquor from the control reactor hosted more Delta/espsilonproteobacteria, Bacteroidetes/Chlorobi, and Spirochaetes.  The absence of filamentous species (Saprospiraceae) in the sludge samples from the ozone-treated reactor was consistent with the microscopic observation, which explained the significant improvement of sludge settling. The similar diversity and fraction of the nitrifiers in the control reactor and the ozonated one explains that nitrification was not affected when mixed liquor ozonation was combined with an EBPR system. About 0.32% of the gene tags were identified as nitrifiers in the control reactor, while about 0.38% were similarly identified in the ozonated system. The major PAOs Candidatus Accumulibacter sp. and Candidatus Halomonas phosphatis were identified in mixed liquor samples from both reactors. It was found that a higher percentage of the species Candidatus Accumulibacter sp. occurred in the samples from the control reactor (2.12%) than in the samples from the ozone-treated one (0.83%); while a significantly higher number of the species Candidatus Halomonas phosphatis was found in the samples (69 tags) from the ozone-treated reactor than those (7 tags) from the control.  Overall the sludge samples from the ozone-treated SBR exhibited lower diversity than those from the control reactor and from the UBC Pilot plant.  • Mixed Liquor Ozonation The efficiency of mixed liquor ozonation depends on TSS/VSS concentration, sludge type, the presence of carbonates and bicarbonates, and the pH.  170 The mixed liquor ozonation with a low TSS concentration achieved a high degree of sludge solubilisation and high cellular inactivation efficiencies. There was an optimum TSS/VSS concentration at which the maximum soluble COD was achieved. The type of sludge had a significant impact on sludge solubilisation and cellular inactivation during mixed liquor ozonation. Both the particle size distribution and the structure of flocs had significant impacts on the performance of sludge solubilisation.  Carbonate/bicarbonate addition may reduce the efficiency of sludge disintegration. The solids solubilization was significantly improved under high pH conditions. Under low pH conditions, more than 90% cellular inactivation was achieved. The mixed liquor from the ozonated reactor exhibited more resistance to ozone treatment at high pH than at low pH.  • Zero Sludge EBPR system A zero sludge EBPR SBR process coupled with an ozone-treatment unit and the phosphorus release and discharge process was operated for 136 days. The optimum ozone dosage was about 70 s per cycle (375 mg O3/day) to maintain a dynamic balance of suspended solids in the system without sludge wasting. The VSS/TSS ratio was not affected and no inorganic matter accumulation was observed. The COD removal, nitrification and phosphorus removal efficiencies were not affected.  By using a batch P-release and discharge approach with VFA addition once every 1 to 2 weeks, the system maintained P mass balance and no P accumulation in the reactor was observed, which indicates that a zero sludge EBPR system would be feasible.  171 8.3 Engineering Significance The conventional excess sludge handling and treatment processes in wastewater treatment plants involve thickening, stabilization, conditioning and dewatering processes, which require huge amounts of electricity and chemicals, constituting about half of the overall costs of wastewater treatment. The present study developed an approach to reduce sludge yield and to minimize sludge production in an EBPR system by combining long SRT operation with mixed liquor ozone treatment.  The following is the list of significant breakthroughs brought about the study. • Significant sludge reduction. Coupling mixed liquor ozonation with a long SRT operation can reduce sludge production by 80% in an EBPR system. • Good effluent quality. The low-dosage mixed liquor ozone treatment applied to an EBPR process may not affect the effluent quality. • Improved sludge settling properties. The sludge settling properties were significantly improved with ozonation due to the absence of filamentous species in the mixed liquor, which can support high sludge concentrations and longer SRT. The improved sludge settling properties would also increase the capacity of a secondary clarifier if gravity separation of liquid and particulates is used . • The similar nitrification performance. The specific ammonium removal rate was high and nitrification was not affected in the ozonated reactor.  • Good phosphorus removal performance. The maximum P-release rate and P-uptake rate from the ozonated reactor were higher than or similar to that of the control. 172 • High fraction of active organisms. The mixed liquor in the ozonated reactor hosted a similiar fraction of active organisms to that of the control. There was a different composition of the bacterial community in the ozonated reactor and the control. An overall lower diversity of the microbial community was observed in the ozonated reactor. • The improvement of mixed liquor ozonation. There was an optimum TSS/VSS concentration at which the maximum soluble COD can be achieved. Carbonate/bicarbonate may reduce the efficiency of sludge disintegration. The solids solubilization was significantly improved under high pH conditions. • Zero Sludge EBPR system. Coupled with an ozone-treatment unit and the phosphorus release and discharge process, a zero EBPR system can be achieved.  8.4 Future Research The present study generated new research objectives that need to be addressed in the future, which are listed as below. • It should be demonstrated that significant sludge reduction can be achieved in a pilot-scale or full-scale EBPR system coupled with mixed liquor ozonation and long SRT operation. • A zero excess sludge EBPR system coupled with mixed liquor ozonation and P release and recovery should be further verified at pilot-scale. • The removal of emerging contaminants in an EBPR system coupled with a mixed liquor ozone treatment unit should be investigated. • The  impact of mixed liquor ozonation on membrane fouling in a MEBPR system should be assessed • The investment and operational costs for sludge reduction by mixed liquor ozonation should be estimated.  173 Bibliography  Adnan, A. (2003). 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0.11 8-Jul-15 326 12 14 40.4 0.05 0.04 0.09 5.14 3.19 2.75 0.06 0.04 9-Jul-15 274 25 2 40.8 0.04 0.01 0.00 4.95 3.08 2.85 0.12 0.07 10-Jul-15 339 13 31 40.1 0.04 0.00 0.00 6.15 5.04 2.97 0.02 0.02 11-Jul-15 329 7 6 43.3 0.01 0.00 0.00 7.33 6.07 3.22 0.07 0.05 12-Jul-15 384 15 13 42.6 0.01 0.06 0.00 8.35 7.89 3.00 0.04 0.05 13-Jul-15 336 30 22 43.5 0.00 0.00 0.00 8.55 8.11 3.06 0.04 0.06 14-Jul-15 341 48 22 41.1 0.03 0.04 0.00 9.11 8.41 3.35 0.08 0.09 15-Jul-15 344 26 39 47.8 0.03 0.01 0.00 8.77 8.92 3.60 0.07 0.08 16-Jul-15 329 10 18 37.1 0.02 0.01 0.00 5.61 7.63 3.39 0.16 0.14 17-Jul-15 361 13 4 37.9 0.02 0.00 0.00 3.48 7.50 2.84 0.11 0.09 18-Jul-15 311 21 17 44 0.04 0.02 0.00 2.78 7.26 3.04 0.06 0.10 19-Jul-15 309 52 25 46 0.02 0.05 0.00 1.91 6.83 3.08 0.33 0.12 188 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 20-Jul-15 306 22 29 41 0.06 0.05 0.00 1.29 6.88 3.13 0.17 0.16 21-Jul-15 346 29 37 44.7 0.02 0.12 0.00 1.95 6.49 3.37 0.08 0.09 22-Jul-15 371 5 16 38.1 0.03 0.03 0.00 2.43 5.96 3.41 0.09 0.08 23-Jul-15 376 5 36 37.7 0.06 0.06 0.00 2.66 5.55 2.74 0.13 0.08 24-Jul-15 306 3 40 37 0.00 0.04 0.00 2.67 5.84 2.54 0.06 0.05 25-Jul-15 311 2 73 39.2 0.01 0.05 0.00 2.94 6.53 2.91 0.04 0.03 26-Jul-15 321 12 77 35 0.04 0.06 0.01 3.43 7.29 2.98 0.02 0.02 27-Jul-15 311 0 0 41.2 0.01 0.08 0.18 3.59 7.29 3.03 0.05 0.01 28-Jul-15 351 3 93 39.7 0.00 0.02 0.08 3.27 7.02 3.37 0.02 0.02 29-Jul-15 521 0 102 35.6 0.08 0.05 0.02 2.88 7.11 3.24 0.00 0.03 30-Jul-15 364 0 68 38.6 0.02 0.07 0.00 2.33 7.30 0.28 0.05 0.06 31-Jul-15 366 22 30 37.6 0.00 0.98 0.00 1.58 7.34 0.32 0.09 0.48 1-Aug-15 394 4 15 39 0.02 2.64 0.00 1.13 6.76 0.53 0.07 0.38 2-Aug-15 346 6 26 40.1 0.02 2.95 0.00 0.91 6.82 0.78 0.09 0.35 3-Aug-15 309 7 20 38.1 0.05 2.18 0.00 1.06 7.33 2.00 0.12 0.42 4-Aug-15 344 13 14 39.4 0.06 0.63 -0.02 1.20 7.82 2.73 0.14 0.60 5-Aug-15 301 1 3 36.9 0.13 0.10 0.00 1.41 7.84 2.77 0.16 0.29 6-Aug-15 271 0 9 37.2 0.03 0.02 0.00 1.79 7.65 2.83 0.13 0.21 7-Aug-15 244 0 3 35.8 0.04 0.02 0.00 2.13 7.35 2.85 0.05 0.15 8-Aug-15 241 0 0 41.3 0.03 0.00 0.00 2.01 7.62 2.81 0.03 0.19 9-Aug-15 244 0 22 42.1 0.04 0.13 0.00 1.52 7.97 2.68 0.02 0.24 10-Aug-15 211 0 0 42.8 0.03 0.06 0.00 0.91 8.32 2.65 0.03 0.22 11-Aug-15 316 32 28 44.2 0.00 0.12 0.06 1.10 9.74 3.24 0.07 0.40 12-Aug-15 304 0 0 43.4 0.03 0.21 0.00 0.79 8.36 2.42 0.15 0.57 13-Aug-15 349 43 49 36.8 0.00 0.16 0.00 0.74 8.75 2.38 0.14 0.67 14-Aug-15 291 60 26 40.1 0.04 0.09 0.00 1.55 9.24 2.66 0.18 0.56 15-Aug-15 369 32 37 38.1 0.04 0.00 0.00 1.55 9.09 2.40 0.14 0.75 189 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 16-Aug-15 434 16 26 39.4 0.01 0.04 0.00 1.15 8.77 2.00 0.13 0.72 17-Aug-15 499 0 16 36.9 0.02 0.17 0.00 0.90 8.09 2.26 0.25 0.58 18-Aug-15 386 0 8 37.2 0.12 0.02 0.00 1.32 7.41 2.22 0.15 0.38 19-Aug-15 569 0 0 35.8 0.94 0.19 0.00 2.62 5.84 2.87 0.08 0.29 20-Aug-15 609 0 0 41.3 0.05 0.00 0.00 2.04 6.29 2.85 0.08  21-Aug-15 416 0 0 42.1 0.03 0.04 0.00 1.36 6.54 2.85 0.05 0.26 22-Aug-15 406 0 0 42.8 0.04 0.09 0.00 1.16 6.48 2.82 0.06 0.27 23-Aug-15 354 0 33 44.2 0.06 0.05 0.00 1.33 6.69 3.20 0.07 0.29 24-Aug-15 354 30 0 43.4 0.03 0.11 0.01 1.80 5.83 2.85 0.06 0.27 25-Aug-15 336 46 33 36.8 0.01 0.01 0.04 2.14 6.20 3.35 0.05 0.27 26-Aug-15 294 35 59 39 0.00 0.00 0.00 2.51 6.46 3.37 0.05 0.25 27-Aug-15 301 21 11 38.5 0.08 0.06 0.02 2.78 6.48 3.36  0.15 28-Aug-15 276 56 73  0.01 0.08 0.03 2.68 6.10 1.76 0.03 0.11 29-Aug-15 389 82 79  0.04 0.37 0.01 1.66 3.99 2.20   30-Aug-15 326 102 81  0.02 0.02 0.00 0.92 2.88 2.37   31-Aug-15 309 16 8  0.00 0.09 0.00 3.25 4.11 1.86   1-Sep-15 259 32 32  0.02 0.32 0.03 3.14 4.86 2.04 0.53  2-Sep-15 356 36 30  0.03 0.09 0.01 3.12 5.14 2.30 0.15 0.43 3-Sep-15 369 89 70  0.07 0.08 0.00 4.84 6.43 2.75 0.05 0.14 4-Sep-15 291 31 39  0.03 0.09 0.01 4.54 7.07 2.76 0.02 0.41 5-Sep-15 351 32 38  0.02 0.02 0.00 4.10 7.18 3.01 0.02 0.06 6-Sep-15 321 35 56  0.04 0.01 0.00 2.82 6.91 2.86 0.11 0.06 7-Sep-15 354 71 24  0.04 0.01 0.00 3.57 7.81 3.13 0.12 0.06 8-Sep-15 401 22 65  0.26 0.08 0.00 3.60 7.65 3.42 0.06 0.09 9-Sep-15 326 78 39  0.36 0.03 0.00 3.59 7.42 3.24 0.07 0.11 10-Sep-15 321 92 37  4.49  0.00   3.11  0.18 11-Sep-15 351 56 52  1.64  0.00   3.24  0.16 190 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 12-Sep-15 314 30 47  0.37 2.23 0.00   3.42  0.22 13-Sep-15 324 26 21  0.32 0.16 0.06 10.40 11.80 3.62 1.00 0.17 14-Sep-15 339 18 0  0.12 0.08 0.00 7.94 9.90 4.20 0.45 0.12 15-Sep-15 351 33 0  0.32 0.16 0.00 4.46 8.31 3.69 0.23 0.13 16-Sep-15 301 20 30 42 0.30 0.12 0.00 3.21 7.92  0.14 0.11 17-Sep-15 254 23 37 49.7 0.15 0.37 0.00 2.73 7.51  0.22 0.10 18-Sep-15 356 32 102 48.3 0.21 0.12 0.00 1.94 6.92  0.11 0.10 19-Sep-15 321 31 36          20-Sep-15 311 27 21          21-Sep-15 341 34 24          22-Sep-15 276 50 102          23-Sep-15 306 51 27          24-Sep-15 379 17 31          25-Sep-15 281 31 32          26-Sep-15 305 35 47          27-Sep-15 329 38 62          28-Sep-15 296 26 75          29-Sep-15 299 34 102          30-Sep-15 319 29 20          1-Oct-15 326 75 72          2-Oct-15 331 53 15          3-Oct-15 334 68 49          4-Oct-15 434 51 54          5-Oct-15 254 53 37          6-Oct-15             7-Oct-15             8-Oct-15             191 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 9-Oct-15             10-Oct-15             11-Oct-15             12-Oct-15             13-Oct-15             14-Oct-15             15-Oct-15             16-Oct-15             17-Oct-15             18-Oct-15             19-Oct-15 314 40 57 43.4 0.13 0.15 0.00 6.22 6.31 3.38 0.07 0.01 20-Oct-15 294 51 53 40.2 0.03 0.04 0.00 6.29 7.88 3.53 0.06 0.07 21-Oct-15 301 35 41 45.2 0.00 0.00 0.00 6.22 7.62 3.19 0.11 0.10 22-Oct-15 261 48 51 44.3 0.01 0.09 0.00 7.22 8.70 3.04 0.10 0.13 23-Oct-15 314 31 33 44.8 0.09 0.12 0.00 7.80 9.08 3.73 0.12 0.13 24-Oct-15 296 39 41 42.8 0.03 0.12 0.00 8.50 9.56 3.24 0.11 0.14 25-Oct-15 349 35 40 46.5 0.07 0.05 0.00 8.50 9.22 3.62 0.08 0.13 26-Oct-15 399 41 43 39.9 0.06 0.00 0.00 8.31 8.79 3.09 0.09 0.13 27-Oct-15 314 48 46 38.5 1.33 1.84 0.00 4.87 5.25 3.56   28-Oct-15 311 51 58 40 2.87 0.71 0.00 3.30 5.26 3.61   29-Oct-15 256 61 59 10.3 0.08 0.00 0.00 6.72 6.47 2.66  0.90 30-Oct-15 284 52 52 21.6 0.01 0.08 0.00 5.59 5.12 2.40  0.54 31-Oct-15 309 43 45 17.4 0.06 0.14 0.00 6.07 4.70 2.00 0.94 0.32 1-Nov-15 254 48 53 22.8 0.03 0.03 0.00 5.08 4.12 2.26 0.15 0.17 2-Nov-15 261 56 73 36.5 0.01 0.04 0.00 5.16 4.80 2.22 0.15 0.12 3-Nov-15 229 57 64 35.5 0.09 0.00 0.00 5.41 5.38 2.87 0.08 0.10 4-Nov-15 246 55 54 30.8 0.07 0.05 0.00 5.55 5.41 2.37 0.12 0.12 192 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 5-Nov-15 149 7 60 30.3 0.02 0.05 0.00 5.07 5.26 2.66 0.10 0.08 6-Nov-15 154 45 56 27.7 0.04 0.08 0.14 5.07 5.68 2.74 0.07 0.07 7-Nov-15 219 33 19 32.1 0.07 0.05 0.00 4.63 4.96 1.56 0.01 0.08 8-Nov-15 209 40 41 34.1 0.13 0.17 0.00 4.12 4.56 1.94 0.03 0.05 9-Nov-15 249 0 0 32.5 0.10 0.10 0.00 5.00 4.86 2.00 0.02 0.04 10-Nov-15 386 44 50 32.2 0.10 0.10 0.00 4.78 4.41 2.55 0.01 0.00 11-Nov-15 264 50 43 35.7 0.10 0.10 0.00 5.02 5.11 2.28 0.01 0.00 12-Nov-15 276 53 37 40.1 0.10 0.10 0.00 5.76 5.29 3.20 0.01 0.00 13-Nov-15 204 52 46 29.1 0.10 0.10 0.00 6.76 6.67 1.92 0.00 0.00 14-Nov-15 224 45 39 27.9 0.10 0.10 0.00 6.98 5.91 1.70 0.00 0.00 15-Nov-15 466 52 45 40.2 0.10 0.10 0.00 7.10 6.37 2.57 0.00 0.00 16-Nov-15 186 82 52 37.1 0.10 0.29 0.00 7.00 7.34 2.09 0.00 0.00 17-Nov-15 144 85 45 36.7 0.33 0.17 0.00 3.85 2.93 2.39 0.00 0.02 18-Nov-15 276 54 19 46.9 0.10 0.10 0.00 3.19 2.95 2.73 0.24 0.05 19-Nov-15 376 31 44 36.2 0.10 0.10 0.00 4.15 4.12  0.02 0.03 20-Nov-15 414 36 34 41.5 0.10 0.10 0.08   1.99   21-Nov-15 409 33 45 39.9 0.10 0.10 0.00   2.51   22-Nov-15 409 119 164 33.5 0.10 0.10 0.04   2.41   23-Nov-15 306 30 36 31 0.10 0.10 0.00   2.80   24-Nov-15 346 34 31 37 0.10 0.10 0.00   2.59   25-Nov-15 304 58 61 42.9 0.01 0.02 0.03 6.88 7.27 3.00 0.01 0.01 26-Nov-15 356 0 0 45 1.16 0.66 0.00 8.28 7.89 2.78 0.00 0.02 27-Nov-15 369 36 34 50.4 0.04 0.45 0.00 8.10 7.91 3.21 0.00 0.00 28-Nov-15 416 21 34 43.2 0.37  0.00 7.98 1.14 3.14 0.01 0.32 29-Nov-15 314 50 25 49 0.19 1.53 0.00 8.37 7.03 3.27 0.00 0.01 30-Nov-15 406 40 26 44.3 0.23 3.44 0.00 8.54 6.67 3.03 0.01 0.01 1-Dec-15 336 0 39 37.9 0.36 4.79 0.07 7.68 6.43 2.94 0.00 0.00 193 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 2-Dec-15 324 41 31  0.21 8.59  7.30 4.52  0.03 0.03 3-Dec-15 294 0 18 33.6 1.20  0.09 6.75 0.19 2.14 0.00  4-Dec-15 319 27 9 33.2 2.08 0.59 0.03 7.28 4.68 2.47 0.01 0.17 5-Dec-15 259 13 16 33.4 0.84  0.00 7.45 3.77 2.39 0.18 0.02 6-Dec-15 191 25 31 22.9 1.83 8.55 0.00 7.70 8.07 1.79 0.86 0.03 7-Dec-15 196 17 1 24.7 0.90 1.72 0.00 6.81 9.47 1.95 0.50 0.02 8-Dec-15 219 27 15 24.3 0.00 0.02 0.06 5.94 6.82 1.77 0.13 0.02 9-Dec-15 164 27 39 31.1 0.22 0.00 0.06 2.83 6.09 1.72 0.00 0.03 10-Dec-15 201 26 17 35.5 0.42 0.00 0.00 2.57 6.40 24.40 0.07 0.04 11-Dec-15 166 44 38 42.1 0.00 0.01 0.18 4.83 7.07 12.30 0.05 0.03 12-Dec-15 324 0 37 42.9 0.01 0.00 0.00 5.91 7.77 2.25 0.05 0.04 13-Dec-15 311 129 118 36 0.00 0.33 0.00 6.74 3.81 2.43 0.04 0.00 14-Dec-15 171 38 78 37 0.00 0.00 0.05 7.54 7.17 2.79 0.04 0.08 15-Dec-15 246 115 125  0.10 0.10  5.59 7.00  0.00 0.00 16-Dec-15 406 103 110 36.1 0.10 0.10 0.00 7.58 7.58 2.20 0.10 0.04 17-Dec-15 424 98 101 38.3 0.10 0.10 0.00 6.74 7.57 2.22  0.02 18-Dec-15 273 62 62 31.6 0.10 0.10 0.00 4.70 6.41 1.79  0.01 19-Dec-15 226 69 69 29.3 0.10 0.10 0.00 4.29 6.29 1.87 0.58 0.00 20-Dec-15 324 64 189 29.5 0.10 2.55 0.00 4.34 4.34 2.05 0.19 0.00 21-Dec-15 169 62 280 36.2 0.10 1.68 0.00 4.64 7.90 2.47 0.06 0.00 22-Dec-15 299 96 70 29.4 0.10 0.10 0.11 5.28 7.51 2.37 0.07 0.03 23-Dec-15 284 48 33 29.8 0.10 0.10 0.00 4.09 7.22 2.03 0.06 0.02 24-Dec-15 199 25 21 29.2 4.88 0.10 0.00 2.33 6.24 1.37 0.06 0.04 25-Dec-15 289 0 27 31.5 3.59 0.10 0.00 7.22 5.76 2.06 0.09 0.03 26-Dec-15 281 43 59 38 0.10 0.10 0.03 6.87 6.21 2.31 0.04 0.03 27-Dec-15 251 45 20 35.5 0.10 0.10 0.13 7.53 6.94 2.27 0.15 0.02 28-Dec-15 256 54 49 32.7 0.10 0.10 0.03 6.35 6.73 2.25 0.12 0.02 194 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 29-Dec-15 299 23 30 37.7 0.10 0.10 0.02 5.72 6.41 2.41 0.10 0.03 30-Dec-15 284 22 27 35.8 0.10 0.10 0.00 5.64 6.76 2.24 0.12 0.00 31-Dec-15 299 24 63 38.7 0.10 0.10 0.03 5.56 6.74 2.53 0.08 0.01 1-Jan-16 271 35 33 44.8 0.10 0.10 0.00 5.38 6.88 2.80 0.04 0.02 2-Jan-16 371 34 42 45 0.10 0.10 0.00 5.77 7.07 2.73 0.06 0.02 3-Jan-16 261 19 11 43.6 0.10 0.10 0.12 6.32 7.00 2.74 0.04 0.02 4-Jan-16 281 36 29 45.4 0.10 0.10 0.00 6.11 7.19 2.65 0.04 0.02 5-Jan-16 471 18 28 44.6 0.10 0.10 0.06 7.19 7.67 2.75 0.08 0.13 6-Jan-16 376 29 19 49 0.10 0.10 0.00   2.62   7-Jan-16 281 40 9 49.9 0.39 0.12 0.08 9.82 9.62 2.67 0.02 0.18 8-Jan-16 284 29 19 48.5 0.50 0.12 0.04 9.42 9.18 2.84 0.00 0.03 9-Jan-16 319 36 62 47.6 0.10 0.31 0.00 5.85 9.18 2.89 0.03 0.00 10-Jan-16 314 29 11 49.7 0.04 0.37 0.00 9.19 9.78 3.20 0.01 0.06 11-Jan-16 331 39 32 37.2 0.32 0.66 0.08 9.64 9.53 2.91 0.01 0.01 12-Jan-16 341 53 16 37.7 0.36 0.28 0.04 10.20  2.58 0.01 0.10 13-Jan-16 299 32 58 25.8 0.23 7.00 0.08 12.80 5.22 2.60 0.01  14-Jan-16 254 25  40.3 0.20 2.95 0.03  5.84 2.18 0.10  15-Jan-16 284 16  39.4 0.06 0.52 0.05  6.98 2.42 0.45  16-Jan-16 259 87 52 38 0.05 0.10 0.00 12.10 8.60 2.35 0.15 0.56 17-Jan-16 241 37 27 29.6 0.02 0.03 0.04 5.72 9.48 2.38 0.05 0.11 18-Jan-16 214 19 15 30 0.13 0.07 0.05 5.57 8.21 1.59 0.51 0.10 19-Jan-16 261 62 60 33.3 0.03 0.06 0.04 6.68 7.63 2.19 0.63 0.53 20-Jan-16 264 6 31 33.7 0.07 0.11 0.00 8.86 7.72 1.68 0.53 0.17 21-Jan-16 284 49 27 33.7 0.09 0.11 0.00 7.46 8.21 2.12 0.65 0.17 22-Jan-16 199 57 62 27.3 0.01 0.05 0.00 8.89 8.28 1.81 0.27 0.10 23-Jan-16 259 30 41 31.24 0.01 0.07 0.05 8.02 7.97 1.89 0.12 0.08 24-Jan-16    31 0.01 0.04 0.03 6.69 7.27 2.05 0.13 0.07 195 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 25-Jan-16    32.4   0.08   2.08   26-Jan-16             27-Jan-16 256 26 21 30.9 0.10 0.10 0.08 4.57 4.59 2.05 0.17 0.04 28-Jan-16 189 32 43 31.3 0.10 0.10 0.00 5.62 5.97 2.07 0.17 0.06 29-Jan-16 226 22 52 37 0.10 0.10 0.07 5.73 8.00 2.32 0.14 0.10 30-Jan-16 196 29 36 26.1 0.10 0.10 0.00 6.47 10.50 1.84 0.15 0.17 31-Jan-16 269 28 27 35.7 0.10 0.10 0.00 6.78 10.30 2.26 0.14 0.11 1-Feb-16 249 27 16 34.9 0.10 0.10 0.04 7.07 12.30 2.38 0.14 0.11 2-Feb-16 274 49 63 34.8 0.10 0.10 0.06 7.33 12.90 2.10 0.19 0.09 3-Feb-16 249 46 35 36.9 0.10 0.10 0.08 7.32 13.60 2.28 0.05 0.08 4-Feb-16 239 21 32 35.7 0.10 0.10 0.07 7.55 10.80 2.09 0.04 0.07 5-Feb-16 244 0 18 41.5 0.10 0.10 0.02 7.80 8.77 3.02 0.02 0.07 6-Feb-16 251 10 9 37.7 0.10 0.10 0.02 8.17 8.45 2.04 0.02 0.10 7-Feb-16 264 9 26 40.2 0.10 0.10 0.05 11.00 9.59 2.22 0.07 0.27 8-Feb-16 269 26 14 44 0.10 0.10 0.04 12.50 10.10 2.52 0.06 0.36 9-Feb-16 489 16 34 42.6 0.10 0.10 0.05 12.00 9.82 2.66 0.09 0.18 10-Feb-16 319 8 11 44.7 0.10 0.10 0.06 11.20 9.80 2.55 0.10 0.73 11-Feb-16 301 8 18 37.1 0.10 0.10 0.06 10.60 9.97 2.50 0.00  12-Feb-16 231 12 11 33.8 0.10 0.10 0.08 9.89 11.10 2.12 0.61  13-Feb-16 446 28 31 31.2 0.10 0.10 0.00 7.14 6.10 1.94   14-Feb-16 311 20 21 28 0.10 0.10 0.16 8.37 6.76 3.28 0.51 0.83 15-Feb-16 231 1 19 32.3 0.10 0.10 0.07 7.74 7.79 0.76 0.38 0.38 16-Feb-16 209 5 20 28.9 1.79 2.38 0.00   1.83 0.43 0.36 17-Feb-16 339 28 23 36.6 0.18 0.40 0.06   1.30 0.19 0.22 18-Feb-16 289 9 18 29.3 0.03 0.03 0.05 9.10 9.59 1.81 0.11 0.09 19-Feb-16 346 56 45 31.5 0.00 0.01 0.10 9.07 8.37 0.83 0.05 0.04 20-Feb-16 209 19 16 30.1 0.01 0.01 0.07 7.75 7.94 1.57 0.00 0.03 196 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 21-Feb-16 286 19 21 32.2 0.01 0.02 0.09 7.28 8.30 1.41 0.00 0.19 22-Feb-16 286 36 28 34.3 0.00 0.01 0.11 7.68 8.54 1.24 0.00 0.01 23-Feb-16 429 3 15 33.9 0.00 0.02 0.00 8.22 9.19 1.40 0.00 0.02 24-Feb-16 574 25 20 38.9 0.00 0.02 0.07 8.68 9.49 2.31 0.00 0.02 25-Feb-16 346 6 11 37.8 0.00 0.02 0.02 8.99 9.96 2.09 0.03 0.04 26-Feb-16 264 20 8 42 0.01 0.00 0.06 9.38 10.40 1.61 0.00 0.06 27-Feb-16 281 12 12 40.7 0.00 0.01 0.00 9.42 10.40 1.96 0.02 0.07 28-Feb-16 269 9 16 43.6 0.00 0.00 0.02 9.30 10.20 2.09 0.03 0.09 29-Feb-16 309 5 13 42.7 0.63 0.65 0.06 8.27 8.17 2.41 0.12 0.00 1-Mar-16 299 61 49  0.12 0.05 0.00 8.41 9.28  0.00 0.02 2-Mar-16 236 0 18  0.15 0.08 0.00 7.52 8.83  0.00 0.00 3-Mar-16 246 27 17  0.04 0.01 0.00 7.15 8.00  0.00 0.01 4-Mar-16 331 4 15 32.5 0.02 0.00 0.00 7.63 7.20 1.82 0.08 0.10 5-Mar-16 259 0 22 35.1 0.28 0.21 0.00 7.42 7.46 1.71 0.01 0.02 6-Mar-16 256 11 9 37.1 0.03 0.05 0.00 7.64 7.24 1.89 0.02 0.02 7-Mar-16 239 1 16 35.5 0.08 0.08 0.00 7.54 7.45 1.70 0.01 0.17 8-Mar-16 231 11 12 38.1 0.03 0.07 0.00   2.68 0.23 0.09 9-Mar-16 281 9 0 30.3 0.02 0.03 0.00   1.99 0.01 0.17 10-Mar-16 216 0 0 38.4 0.03 0.05 0.00 10.30 9.90 2.26 0.01 0.00 11-Mar-16 276 13 33 38.2 0.15 0.13 0.00 8.82 7.64 2.20 0.00 0.07 12-Mar-16 251 35 23 34.9 0.02 0.05 0.00 12.00 7.25 1.99 0.01 0.14 13-Mar-16 239 8 15 37.6 0.02 0.01 0.00 12.30 7.06 1.97 0.28 0.09 14-Mar-16 301 10 9 38.6 0.07 0.03 0.00 12.90 6.94 2.28 0.11 0.11 15-Mar-16 321 5 27 41.2 0.02 0.03 0.00 9.16 6.31 5.76 0.10 0.16 16-Mar-16 276 10 8 36.3 0.03 0.02 0.00 7.02 6.60 3.95 0.11 0.22 17-Mar-16 384 8 18 31.4 0.05 0.02 0.06 7.17 6.60 2.14 0.09 0.26 18-Mar-16 401 9 16 34.8 0.01 0.04 0.00 5.16 5.35 2.30 0.49  197 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 19-Mar-16 356 16 22 36.7 0.04 0.02 0.00 7.01 6.57 1.79 0.12 0.76 20-Mar-16 339 35 20 37.8 0.98 0.00 0.00 7.30 6.89 2.66 0.14 0.45 21-Mar-16 274 27 33 36.4 0.01 0.06 0.00 8.04 7.69 2.13 0.15 0.55 22-Mar-16 286 15 23 37.3 0.03 0.06 0.00 7.90 7.72 4.61 0.17  23-Mar-16 364 13 18 39.7 0.02 0.08 0.00 7.38 7.74 2.49 0.02 0.29 24-Mar-16 294 22 32 42.6 0.01 0.05 0.00 8.00 7.98 4.92 0.22 0.26 25-Mar-16 296 18 45 43.2 0.05 0.05 0.00 7.53 7.49 2.04 0.11 0.22 26-Mar-16 261 36 22 43.5 0.07 0.07 0.00 7.42 7.51 2.72 0.09 0.24 27-Mar-16 264 10 26 37.9 0.01 0.03 0.00 7.31 7.95 1.83 0.01 0.25 28-Mar-16 319 6 13 42.2 0.01 0.04 0.00 6.98 6.93 2.44 0.08 0.23 29-Mar-16 191 18 19 32.7 0.11 0.03 0.00 6.79 6.94 2.35 0.14 0.19 30-Mar-16 314 12 14 37.8 0.04 0.05 0.00 7.05 6.96 2.33 0.09 0.21 31-Mar-16 359 6 20 37.5 0.01 0.05 0.00 7.29 7.04  0.05 0.10 1-Apr-16 304 6 31 38.5 0.11 0.03 0.00 7.56 7.15  0.03 0.07 2-Apr-16 299 19 19 35.8 0.05 0.09 0.00 5.85 5.37  0.08 0.07 3-Apr-16 294 14 19 38.3 0.03 0.04 0.00 7.91 7.22  0.14 0.07 4-Apr-16 361 26 17 42.7 0.02 0.05 0.00 8.47 7.83  0.13 0.05 5-Apr-16 269 22 15 39.5 0.02 0.01 0.00 8.55 9.26  0.11 0.07 6-Apr-16 316 21 15 41.6 0.02 0.05 0.00 7.98 12.40  0.11 0.03 7-Apr-16 339 11 9 37.8 0.03 0.00 0.00 8.46 11.80  0.06 0.02 8-Apr-16 306 18 25 47.7 0.04 0.04 0.00 9.31 9.93  0.13 0.11 9-Apr-16 319 46 23 45.9 0.08 0.06 0.00 9.95 9.74  0.30 0.15 10-Apr-16 301 11 18 47.9 0.05 0.07 0.00 9.91 9.49  0.22 0.14 11-Apr-16 284 13 15 46.3 1.67 2.12 0.00 7.69 7.16    12-Apr-16 291 8 19 45.1 1.04 4.68 0.00 11.00 8.51    13-Apr-16 304 17 39 41.3 0.09 3.94 0.04 10.80 12.50 0.78   14-Apr-16 311 15 23 42.2 0.10 0.80 0.00 10.30 12.40 2.80 0.53  198 Date Total COD NH4+-N NOx-N PO4-P Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated Influent The control The ozonated 15-Apr-16 294 20 15 43.7 0.04 0.84 0.04 9.83 10.60 2.73 0.33 0.98 16-Apr-16 336 40 21 45 0.07 0.70 0.00 9.58 10.20 2.15 0.20 0.68 17-Apr-16 326 6 11 45.8 0.01 0.89 0.04 9.53 10.10 2.64 0.14  18-Apr-16 306 11 11 48.1 0.01 3.76 0.00 9.89 8.20 2.47 0.24  19-Apr-16 329 0 0 45 0.01 10.00 0.05 8.98 4.18 2.30 0.33  20-Apr-16 301 0 17 43.8 0.04 3.34 0.00 10.10 10.70 2.89 0.61  21-Apr-16 294 0 11 33.8 0.11 0.09 0.07 10.40 11.10 2.52 0.74  22-Apr-16 234 2 26 36.3 0.07 0.05 0.02 10.10 10.50 2.84 0.83 0.98 23-Apr-16 316 0 0 36.4 0.10 0.07 0.05 10.20 10.20 2.76 0.87 0.28 24-Apr-16 334 0 0 36 0.01 0.11 0.05 9.98 10.30 3.10  0.23 25-Apr-16 299 0 0 35.8 0.09 0.05 0.04 9.49 10.10 2.93  0.22 26-Apr-16 279 0 0 35.4 0.07 0.21 0.03 11.40 11.50 2.91  0.21 27-Apr-16 294 1 39 37.5 0.81 0.79 0.56 11.20 11.20 2.50  0.22 28-Apr-16 311 0 25 39.1 0.23 0.16 0.55 9.29 12.30 2.24   29-Apr-16 224 43 0  0.08 0.11 0.52 8.10 7.98 2.25   30-Apr-16     0.04 0.02 0.45 10.70 1.36 2.43  0.50  199 Appendix B  The identified species and their percentages of the samples from the control SBR Finest Classification Taxonomy # % species:uncultured Bacteroidetes bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 4727 13.76% species:uncultured delta proteobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> no rank:environmental samples 4394 12.79% species:uncultured soil bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 4253 12.38% species:uncultured beta proteobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:environmental samples 1927 5.61% species:uncultured planctomycete Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:environmental samples 1755 5.11% species:uncultured Spirochaetes bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Spirochaetes -> class:Spirochaetia -> no rank:environmental samples 1215 3.54% species:uncultured Verrucomicrobia bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> no rank:environmental samples 1030 3.00% species:uncultured alpha proteobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 961 2.80% species:uncultured Candidatus Accumulibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:unclassified Betaproteobacteria -> genus:Candidatus Accumulibacter -> no rank:environmental samples 728 2.12% species:uncultured gamma proteobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> no rank:environmental samples 706 2.05% species:uncultured Chloroflexi bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> no rank:environmental samples 656 1.91% species:uncultured actinobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> no rank:environmental samples 537 1.56% species:uncultured Acidobacteria bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Fibrobacteres/Acidobacteria group -> phylum:Acidobacteria -> no rank:environmental samples 508 1.48% 200 Finest Classification Taxonomy # % species:uncultured Rubrobacterales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Rubrobacteria -> order:Rubrobacterales -> no rank:environmental samples 374 1.09% species:uncultured sludge bacterium H2 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> no rank:environmental samples 293 0.85% species:uncultured Rhizobiales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> no rank:environmental samples 281 0.82% species:uncultured Gemmatimonadetes bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Gemmatimonadetes -> no rank:environmental samples 252 0.73% species:uncultured bacterium PHOS-HE21 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 224 0.65% species:uncultured Sphingobacteriales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> no rank:environmental samples 223 0.65% species:uncultured Burkholderiales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> no rank:environmental samples 220 0.64% species:uncultured Cytophaga sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Cytophagaceae -> genus:Cytophaga -> no rank:environmental samples 219 0.64% species:Prosthecobacter fluviatilis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobiaceae -> genus:Prosthecobacter 218 0.63% species:uncultured Caldilinea sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Caldilineae -> order:Caldilineales -> family:Caldilineaceae -> genus:Caldilinea -> no rank:environmental samples 204 0.59% species:uncultured bacterium MK09 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 195 0.57% species:Prosthecobacter vanneervenii Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobiaceae -> genus:Prosthecobacter 185 0.54% 201 Finest Classification Taxonomy # % species:uncultured verrucomicrobium DEV006 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> no rank:environmental samples 178 0.52% species:bacterium GPB6 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 173 0.50% species:uncultured Planctomycetales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:environmental samples 156 0.45% species:uncultured sludge bacterium S36 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 146 0.42% species:uncultured Chlorobi bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Chlorobi -> no rank:environmental samples 143 0.42% species:uncultured rumen bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 139 0.40% species:uncultured Comamonadaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> no rank:environmental samples 138 0.40% species:uncultured Rhodospirillales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> no rank:environmental samples 130 0.38% species:uncultured Acetanaerobacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Ruminococcaceae -> genus:Acetanaerobacterium -> no rank:environmental samples 126 0.37% species:uncultured Gemmata sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> family:Planctomycetaceae -> genus:Gemmata -> no rank:environmental samples 121 0.35% species:uncultured proteobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> no rank:environmental samples 120 0.35% species:uncultured Stigmatella sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Cystobacterineae -> family:Cystobacteraceae -> genus:Stigmatella -> no rank:environmental samples 119 0.35% species:agricultural soil bacterium SC-I-12 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples -> no rank:agricultural soil bacteria ensemble 113 0.33% genus:Aetherobacter Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> 109 0.32% 202 Finest Classification Taxonomy # % suborder:Sorangiineae -> family:Polyangiaceae species:uncultured marine bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 106 0.31% species:uncultured sludge bacterium A17 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:environmental samples 98 0.29% species:uncultured Achromobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Alcaligenaceae -> genus:Achromobacter -> no rank:environmental samples 97 0.28% species:uncultured Haliscomenobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Saprospiraceae -> genus:Haliscomenobacter -> no rank:environmental samples 95 0.28% species:uncultured bacterium mle1-27 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> no rank:environmental samples 91 0.26% species:uncultured Clostridiales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> no rank:environmental samples 91 0.26% species:Curvibacter putative symbiont of Hydra magnipapillata Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Curvibacter 88 0.26% species:uncultured deep-sea bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 82 0.24% species:uncultured Armatimonadetes bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Armatimonadetes -> no rank:environmental samples 81 0.24% species:uncultured compost bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 74 0.22% species:uncultured Acidobacteriales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Fibrobacteres/Acidobacteria group -> phylum:Acidobacteria -> class:Acidobacteriia -> order:Acidobacteriales -> no rank:environmental samples 73 0.21% species:uncultured Bacteroidales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> no rank:environmental samples 73 0.21% species:uncultured Bacteroides sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> 72 0.21% 203 Finest Classification Taxonomy # % order:Bacteroidales -> family:Bacteroidaceae -> genus:Bacteroides -> no rank:environmental samples species:uncultured Planctomycetaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> family:Planctomycetaceae -> no rank:environmental samples 65 0.19% species:uncultured Rhodocyclaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> no rank:environmental samples 64 0.19% species:uncultured sludge bacterium A27b Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 63 0.18% species:uncultured Brochothrix sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Listeriaceae -> genus:Brochothrix -> no rank:environmental samples 59 0.17% species:uncultured Burkholderia sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Burkholderia -> no rank:environmental samples 58 0.17% species:uncultured sludge bacterium H8 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:environmental samples 57 0.17% species:uncultured Prosthecobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobiaceae -> genus:Prosthecobacter -> no rank:environmental samples 52 0.15% species:uncultured Phycisphaerales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Phycisphaerae -> order:Phycisphaerales -> no rank:environmental samples 52 0.15% species:uncultured Verminephrobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Verminephrobacter -> no rank:environmental samples 47 0.14% species:uncultured Hyphomicrobiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Hyphomicrobiaceae -> no rank:environmental samples 46 0.13% species:uncultured Bdellovibrio sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Bdellovibrionales -> family:Bdellovibrionaceae -> genus:Bdellovibrio -> no rank:environmental samples 45 0.13% 204 Finest Classification Taxonomy # % species:uncultured Termite group 1 bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Elusimicrobia -> no rank:environmental samples 45 0.13% species:Myxococcales bacterium enrichment culture clone 11380 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> no rank:environmental samples 44 0.13% species:Cytophaga sp. BHI60-57B Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Cytophagaceae -> genus:Cytophaga 43 0.13% species:Stella humosa Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> family:Acetobacteraceae -> genus:Stella 39 0.11% species:uncultured bacterium PHOS-HE28 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 38 0.11% species:Comamonas denitrificans Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Comamonas 37 0.11% species:uncultured Sphingobacteria bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> no rank:environmental samples 37 0.11% species:uncultured Nitrospira sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Nitrospirae -> class:Nitrospira -> order:Nitrospirales -> family:Nitrospiraceae -> genus:Nitrospira -> no rank:environmental samples 37 0.11% species:Pelomonas puraquae Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Pelomonas 34 0.10% species:Arenimonas aquatica Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Arenimonas 34 0.10% species:uncultured Bradyrhizobium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Bradyrhizobiaceae -> genus:Bradyrhizobium -> no rank:environmental samples 32 0.09% species:uncultured bacterium PHOS-HE25 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 30 0.09% 205 Finest Classification Taxonomy # % species:uncultured Dechloromonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Dechloromonas -> no rank:environmental samples 28 0.08% species:uncultured Gemmatimonadaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Gemmatimonadetes -> class:Gemmatimonadetes -> order:Gemmatimonadales -> family:Gemmatimonadaceae -> no rank:environmental samples 28 0.08% species:Candidatus Hepatincola porcellionum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rickettsiales -> no rank:Rickettsiales genera incertae sedis -> genus:Candidatus Hepatincola 27 0.08% species:uncultured Acidimicrobidae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Acidimicrobiia -> no rank:environmental samples 27 0.08% species:uncultured Polyangiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Sorangiineae -> family:Polyangiaceae -> no rank:environmental samples 27 0.08% species:uncultured Beijerinckia sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Beijerinckiaceae -> genus:Beijerinckia -> no rank:environmental samples 26 0.08% species:uncultured sludge bacterium H28 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:environmental samples 25 0.07% species:uncultured Firmicutes bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> no rank:environmental samples 25 0.07% genus:Pedomicrobium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Hyphomicrobiaceae 25 0.07% species:Rasbo bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> no rank:unclassified Rhizobiales -> no rank:unclassified Rhizobiales (miscellaneous) 25 0.07% species:uncultured Intrasporangiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Intrasporangiaceae -> no rank:environmental samples 25 0.07% species:Pyrinomonas methylaliphatogenes Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Fibrobacteres/Acidobacteria group -> phylum:Acidobacteria -> no rank:unclassified Acidobacteria -> no rank:Acidobacteria subdivision 4 -> 24 0.07% 206 Finest Classification Taxonomy # % genus:Pyrinomonas species:Candidatus Brownia rhizoecola Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> order:Flavobacteriales -> family:Blattabacteriaceae -> genus:Candidatus Brownia 23 0.07% species:uncultured Thiobacillus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Hydrogenophilales -> family:Hydrogenophilaceae -> genus:Thiobacillus -> no rank:environmental samples 23 0.07% species:Hyphomicrobium sp. Ellin112 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Hyphomicrobiaceae -> genus:Hyphomicrobium 23 0.07% species:filamentous bacterium Plant1 Iso8 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:unclassified Bacteroidetes -> no rank:unclassified Bacteroidetes (miscellaneous) 23 0.07% species:uncultured Caldilineaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Caldilineae -> order:Caldilineales -> family:Caldilineaceae -> no rank:environmental samples 22 0.06% species:uncultured Gallionella sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Gallionellales -> family:Gallionellaceae -> genus:Gallionella -> no rank:environmental samples 21 0.06% species:uncultured Saprospiraceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Saprospiraceae -> no rank:environmental samples 21 0.06% species:uncultured Parvibaculum sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Rhodobiaceae -> genus:Parvibaculum -> no rank:environmental samples 21 0.06% species:uncultured Acidimicrobiales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Acidimicrobiia -> order:Acidimicrobiales -> no rank:environmental samples 21 0.06% species:Streptomyces sp. 14CM003 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Streptomycetales -> family:Streptomycetaceae -> genus:Streptomyces 20 0.06% 207 Finest Classification Taxonomy # % species:uncultured Chitinophagaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Chitinophagaceae -> no rank:environmental samples 20 0.06% species:Rhodobacter sp. enrichment culture clone AOCRB-EC-1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Rhodobacteraceae -> genus:Rhodobacter -> no rank:environmental samples 20 0.06% species:uncultured Opitutae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Opitutae -> no rank:environmental samples 19 0.06% species:uncultured Nitrospirae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Nitrospirae -> no rank:environmental samples 19 0.06% species:Bdellovibrio bacteriovorus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Bdellovibrionales -> family:Bdellovibrionaceae -> genus:Bdellovibrio 19 0.06% species:uncultured Curvibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Curvibacter -> no rank:environmental samples 19 0.06% species:uncultured anaerobic bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 19 0.06% species:halophilic eubacterium EHB-4 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> order:Bacteroidetes Order II. Incertae sedis -> family:Rhodothermaceae -> genus:Salinibacter -> no rank:unclassified Salinibacter 18 0.05% species:gamma proteobacterium HdN1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> no rank:unclassified Gammaproteobacteria -> no rank:unclassified Gammaproteobacteria (miscellaneous) 18 0.05% species:Nitrospira sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Nitrospirae -> class:Nitrospira -> order:Nitrospirales -> family:Nitrospiraceae -> genus:Nitrospira 17 0.05% 208 Finest Classification Taxonomy # % species:uncultured Ilumatobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Acidimicrobiia -> order:Acidimicrobiales -> family:Acidimicrobiaceae -> genus:Ilumatobacter -> no rank:environmental samples 17 0.05% genus:Cellvibrio Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Cellvibrionales -> family:Cellvibrionaceae 17 0.05% species:uncultured Brevundimonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Caulobacterales -> family:Caulobacteraceae -> genus:Brevundimonas -> no rank:environmental samples 16 0.05% species:uncultured halophilic eubacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 16 0.05% species:uncultured delta proteobacterium DeepAnt-32C6 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> no rank:environmental samples 16 0.05% species:beta proteobacterium pACH94 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:unclassified Betaproteobacteria -> no rank:unclassified Betaproteobacteria (miscellaneous) 16 0.05% genus:Nakamurella Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Nakamurellales -> family:Nakamurellaceae 16 0.05% species:uncultured Zoogloea sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Zoogloea -> no rank:environmental samples 15 0.04% species:Brachymonas sp. canine oral taxon 015 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Brachymonas 15 0.04% species:uncultured Flexibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Cytophagaceae -> genus:Flexibacter -> no rank:environmental samples 15 0.04% species:bacterium TG141 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 15 0.04% species:Woodsholea maritima Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Caulobacterales -> family:Caulobacteraceae -> genus:Woodsholea 15 0.04% species:Candidatus Microthrix parvicella Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> no rank:unclassified Actinobacteria -15 0.04% 209 Finest Classification Taxonomy # % > genus:Candidatus Microthrix species:Aminobacter ciceronei Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Phyllobacteriaceae -> genus:Aminobacter 14 0.04% species:Nitrobacter winogradskyi Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Bradyrhizobiaceae -> genus:Nitrobacter 14 0.04% species:uncultured Clostridiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Clostridiaceae -> no rank:environmental samples 14 0.04% species:uncultured Bacteroidetes/Chlorobi group bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> no rank:environmental samples 14 0.04% species:uncultured Acidobacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Fibrobacteres/Acidobacteria group -> phylum:Acidobacteria -> class:Acidobacteriia -> order:Acidobacteriales -> family:Acidobacteriaceae -> genus:Acidobacterium -> no rank:environmental samples 14 0.04% species:uncultured Cystobacterineae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Cystobacterineae -> no rank:environmental samples 14 0.04% species:Maricaulis maris Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Hyphomonadaceae -> genus:Maricaulis 14 0.04% species:drinking water bacterium MB13 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 14 0.04% species:Achromobacter xylosoxidans Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Alcaligenaceae -> genus:Achromobacter 13 0.04% species:uncultured sludge bacterium A7 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 13 0.04% species:uncultured Actinomycetales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Actinomycetales -> no rank:environmental samples 13 0.04% species:uncultured sludge bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 13 0.04% species:bacterium Ellin6529 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 13 0.04% 210 Finest Classification Taxonomy # % species:Planctomycetales bacterium Ellin7244 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:unclassified Planctomycetales -> no rank:unclassified Planctomycetales (miscellaneous) 12 0.03% species:uncultured Flavobacteriia bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> no rank:environmental samples 12 0.03% species:uncultured Chitinophaga sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Chitinophagaceae -> genus:Chitinophaga -> no rank:environmental samples 12 0.03% species:uncultured bacterium PHOS-HC15 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Chlorobi -> no rank:environmental samples 12 0.03% species:Micromonospora sp. 215008 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micromonosporales -> family:Micromonosporaceae -> genus:Micromonospora 12 0.03% species:uncultured Iamia sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Acidimicrobiia -> order:Acidimicrobiales -> family:Iamiaceae -> genus:Iamia -> no rank:environmental samples 12 0.03% species:bacterium WHC2-6 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 12 0.03% species:uncultured Stenotrophomonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Stenotrophomonas -> no rank:environmental samples 12 0.03% species:Paracraurococcus sp. ORS 1473 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> family:Acetobacteraceae -> genus:Paracraurococcus 12 0.03% species:Propionivibrio pelophilus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Propionivibrio 12 0.03% species:Flavobacterium sp. HWG-A1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> order:Flavobacteriales -> family:Flavobacteriaceae -> genus:Flavobacterium 12 0.03% species:uncultured Acinetobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Pseudomonadales -> 11 0.03% 211 Finest Classification Taxonomy # % family:Moraxellaceae -> genus:Acinetobacter -> no rank:environmental samples species:uncultured Crater Lake bacterium CL500-15 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples -> no rank:Crater Lake bacteria ensemble 11 0.03% species:Bacillus subtilis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Bacillaceae -> genus:Bacillus -> species group:Bacillus subtilis group 11 0.03% species:uncultured Flammeovirgaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Flammeovirgaceae -> no rank:environmental samples 11 0.03% species:alpha proteobacterium A0839 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:unclassified Alphaproteobacteria -> no rank:unclassified Alphaproteobacteria (miscellaneous) 11 0.03% species:uncultured Rikenellaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Rikenellaceae -> no rank:environmental samples 11 0.03% order:Opitutales Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Opitutae 11 0.03% species:uncultured Lentisphaerae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Lentisphaerae -> no rank:environmental samples 11 0.03% species:bacterium enrichment culture clone auto123_4W Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 11 0.03% species:uncultured sediment bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 11 0.03% species:uncultured Rhodanobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Rhodanobacter -> no rank:environmental samples 11 0.03% species:uncultured Clostridium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Clostridiaceae -> genus:Clostridium -> no rank:environmental samples 11 0.03% species:uncultured sludge bacterium A26 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 11 0.03% 212 Finest Classification Taxonomy # % species:Delftia sp. RF-93 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Delftia 10 0.03% species:Agrobacterium tumefaciens Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Rhizobiaceae -> no rank:Rhizobium/Agrobacterium group -> genus:Agrobacterium -> species group:Agrobacterium tumefaciens complex 10 0.03% species:Ensifer adhaerens Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Rhizobiaceae -> no rank:Sinorhizobium/Ensifer group -> genus:Ensifer 10 0.03% species:Blastochloris viridis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Hyphomicrobiaceae -> genus:Blastochloris 10 0.03% no rank:Nafulsella turpanensis ZLM-10 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Flammeovirgaceae -> genus:Nafulsella -> species:Nafulsella turpanensis 10 0.03% species:uncultured ammonia-oxidizing bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples -> no rank:ammonia oxidising bacteria ensemble 10 0.03% species:planctomycete MS1316 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> family:Planctomycetaceae -> no rank:unclassified Planctomycetaceae 10 0.03% species:uncultured Desulfuromonadales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Desulfuromonadales -> no rank:environmental samples 10 0.03% species:uncultured Planctomycetia bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> no rank:environmental samples 10 0.03% species:uncultured Burkholderiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> no rank:environmental samples 10 0.03% species:uncultured sludge bacterium A41 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 10 0.03% species:uncultured Prolixibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Prolixibacteraceae -> genus:Prolixibacter -> no rank:environmental samples 10 0.03% 213 Finest Classification Taxonomy # % species:Dermatophilus sp. CCUG 48971 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Dermatophilaceae -> genus:Dermatophilus 10 0.03% species:uncultured Cytophagales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> no rank:environmental samples 9 0.03% species:Herbaspirillum sp. TSO33-2 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Oxalobacteraceae -> genus:Herbaspirillum 9 0.03% species:Chitinophaga sp. BS20 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Chitinophagaceae -> genus:Chitinophaga 9 0.03% no rank:Thioclava pacifica DSM 10166 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Rhodobacteraceae -> genus:Thioclava -> species:Thioclava pacifica 9 0.03% species:uncultured Fibrobacteres bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Fibrobacteres/Acidobacteria group -> phylum:Fibrobacteres -> no rank:environmental samples 9 0.03% species:Bacteroides nordii Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Bacteroidaceae -> genus:Bacteroides 9 0.03% genus:Uliginosibacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae 9 0.03% species:Brevifollis gellanilyticus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobiaceae -> genus:Brevifollis 9 0.03% species:Micavibrio aeruginosavorus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:unclassified Alphaproteobacteria -> genus:Micavibrio 9 0.03% species:uncultured Latescibacteria bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> phylum:Latescibacteria -> no rank:environmental samples 9 0.03% species:uncultured Acidobacteriaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Fibrobacteres/Acidobacteria group -> 9 0.03% 214 Finest Classification Taxonomy # % phylum:Acidobacteria -> class:Acidobacteriia -> order:Acidobacteriales -> family:Acidobacteriaceae -> no rank:environmental samples species:uncultured Flavobacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> order:Flavobacteriales -> family:Flavobacteriaceae -> genus:Flavobacterium -> no rank:environmental samples 9 0.03% species:uncultured Veillonellaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Negativicutes -> order:Selenomonadales -> family:Veillonellaceae -> no rank:environmental samples 9 0.03% genus:Arsenicicoccus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Intrasporangiaceae 9 0.03% species:eubacterium sp. 11-14 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Eubacteriaceae -> genus:Eubacterium 9 0.03% species:Rhizobium leguminosarum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Rhizobiaceae -> no rank:Rhizobium/Agrobacterium group -> genus:Rhizobium 9 0.03% species:uncultured Epsilonproteobacteria bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Epsilonproteobacteria -> no rank:environmental samples 8 0.02% species:Sorghum grassy shoot phytoplasma variant I Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Tenericutes -> class:Mollicutes -> order:Acholeplasmatales -> family:Acholeplasmataceae -> genus:Candidatus Phytoplasma -> no rank:unclassified phytoplasmas 8 0.02% species:Pedobacter sp. NHI-13 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Sphingobacteriaceae -> genus:Pedobacter 8 0.02% species:uncultured Eubacteriaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Eubacteriaceae -> no rank:environmental samples 8 0.02% species:uncultured Deferribacteres bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Deferribacteres -> class:Deferribacteres -> no rank:environmental samples 8 0.02% species:Candidatus Protistobacter heckmanni Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Candidatus Protistobacter 8 0.02% 215 Finest Classification Taxonomy # % species:uncultured Ktedonobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Ktedonobacteria -> order:Ktedonobacterales -> family:Ktedonobacteraceae -> genus:Ktedonobacter -> no rank:environmental samples 8 0.02% species:uncultured bacterium PHOS-HE31 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 8 0.02% species:uncultured Legionella sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Legionellales -> family:Legionellaceae -> genus:Legionella -> no rank:environmental samples 8 0.02% species:uncultured Sphingomonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Sphingomonadales -> family:Sphingomonadaceae -> genus:Sphingomonas -> no rank:environmental samples 8 0.02% species:Clostridium sp. BL8 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Clostridiaceae -> genus:Clostridium 8 0.02% no rank:Paludibacter propionicigenes WB4 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Porphyromonadaceae -> genus:Paludibacter -> species:Paludibacter propionicigenes 8 0.02% species:uncultured Chloroflexus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Chloroflexia -> order:Chloroflexales -> suborder:Chloroflexineae -> family:Chloroflexaceae -> genus:Chloroflexus -> no rank:environmental samples 8 0.02% species:bacterium enrichment culture clone R4-80B Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 8 0.02% species:uncultured Cystobacteraceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Cystobacterineae -> family:Cystobacteraceae -> no rank:environmental samples 7 0.02% species:Paenibacillus sp. XWS-29 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Paenibacillaceae -> genus:Paenibacillus 7 0.02% species:uncultured Flavisolibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Chitinophagaceae -> genus:Flavisolibacter -> no rank:environmental samples 7 0.02% 216 Finest Classification Taxonomy # % species:bacterium WH2-9 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 7 0.02% species:uncultured Singulisphaera sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> family:Planctomycetaceae -> genus:Singulisphaera -> no rank:environmental samples 7 0.02% species:uncultured bacterium PHOS-HE47 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 7 0.02% species:uncultured Nitrosomonadaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Nitrosomonadales -> family:Nitrosomonadaceae -> no rank:environmental samples 7 0.02% species:uncultured Anaerolineaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Anaerolineae -> order:Anaerolineales -> family:Anaerolineaceae -> no rank:environmental samples 7 0.02% species:uncultured Lautropia sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Lautropia -> no rank:environmental samples 7 0.02% species:Catonella sp. canine oral taxon 257 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Lachnospiraceae -> genus:Catonella 7 0.02% species:Serratia marcescens Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Enterobacteriales -> family:Enterobacteriaceae -> genus:Serratia 7 0.02% species:uncultured endolithic bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 7 0.02% species:Shewanella sp. HJ-53 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Alteromonadales -> family:Shewanellaceae -> genus:Shewanella 7 0.02% species:uncultured Acidovorax sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Acidovorax -> no rank:environmental samples 7 0.02% species:uncultured Ralstonia sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Ralstonia -> no rank:environmental samples 7 0.02% species:Streptomyces sp. S7 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Streptomycetales -> family:Streptomycetaceae -> genus:Streptomyces 7 0.02% 217 Finest Classification Taxonomy # % species:Bifidobacterium sp. 113 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Bifidobacteriales -> family:Bifidobacteriaceae -> genus:Bifidobacterium 7 0.02% no rank:Turneriella parva DSM 21527 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Spirochaetes -> class:Spirochaetia -> no rank:Leptospirales -> family:Leptospiraceae -> genus:Turneriella -> species:Turneriella parva 7 0.02% species:Thiobacillus Q Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Hydrogenophilales -> family:Hydrogenophilaceae -> genus:Thiobacillus 7 0.02% species:Caulobacter sp. H62 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Caulobacterales -> family:Caulobacteraceae -> genus:Caulobacter 7 0.02% species:Candidatus Halomonas phosphatis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Oceanospirillales -> family:Halomonadaceae -> genus:Halomonas 7 0.02% species:Candidatus Baumannia cicadellinicola Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> no rank:unclassified Gammaproteobacteria -> no rank:Candidatus Baumannia 7 0.02% species:bacterium str. 37236 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 7 0.02% species:Comamonas sp. ASR12 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Comamonas 6 0.02% species:uncultured bacterium PHOS-HE19 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 6 0.02% species:Kocuria sp. Am 16 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Micrococcaceae -> genus:Kocuria 6 0.02% species:uncultured Bdellovibrionales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Bdellovibrionales -> no rank:environmental samples 6 0.02% species:Bacillus pumilus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Bacillaceae -> genus:Bacillus 6 0.02% species:Lethal yellowing phytoplasma Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Tenericutes -> class:Mollicutes -> order:Acholeplasmatales -> family:Acholeplasmataceae -> genus:Candidatus Phytoplasma -> species group:16SrIV (Coconut lethal yellows group) 6 0.02% 218 Finest Classification Taxonomy # % species:uncultured spirochete Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Spirochaetes -> class:Spirochaetia -> order:Spirochaetales -> no rank:environmental samples 6 0.02% species:uncultured Xanthomonadaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> no rank:environmental samples 6 0.02% species:uncultured Hyphomicrobium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Hyphomicrobiaceae -> genus:Hyphomicrobium -> no rank:environmental samples 6 0.02% species:Rhizobium qilianshanense Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Rhizobiaceae -> no rank:Rhizobium/Agrobacterium group -> genus:Rhizobium 6 0.02% species:uncultured Thiotrichaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Thiotrichales -> family:Thiotrichaceae -> no rank:environmental samples 6 0.02% species:uncultured Xiphinematobacteriaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Spartobacteria -> no rank:environmental samples 6 0.02% species:uncultured Syntrophobacteraceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Syntrophobacterales -> family:Syntrophobacteraceae -> no rank:environmental samples 6 0.02% species:uncultured Rhodospirillaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> family:Rhodospirillaceae -> no rank:environmental samples 6 0.02% species:uncultured Rubrobacteria bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Rubrobacteria -> no rank:environmental samples 6 0.02% species:Chloroflexi bacterium ET1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> no rank:unclassified Chloroflexi -> no rank:unclassified Chloroflexi (miscellaneous) 6 0.02% species:Polynucleobacter cosmopolitanus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Polynucleobacter 6 0.02% species:uncultured bacterium mle1-9 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> no rank:environmental samples 6 0.02% 219 Finest Classification Taxonomy # % species:uncultured Paludibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Porphyromonadaceae -> genus:Paludibacter -> no rank:environmental samples 6 0.02% species:uncultured Ruminococcaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Ruminococcaceae -> no rank:environmental samples 6 0.02% species:uncultured Azoarcus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Azoarcus -> no rank:environmental samples 6 0.02% species:denitrifying bacterium W99 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:unclassified Betaproteobacteria -> no rank:unclassified Betaproteobacteria (miscellaneous) 6 0.02% genus:Hylemonella Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae 6 0.02% genus:Thiocapsa Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Chromatiales -> family:Chromatiaceae 6 0.02% species:uncultured Clostridia bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> no rank:environmental samples 6 0.02% species:uncultured sludge bacterium H34 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 6 0.02% species:bacterium RS5A Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 6 0.02% species:uncultured Aquabacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> no rank:unclassified Burkholderiales -> no rank:Burkholderiales Genera incertae sedis -> genus:Aquabacterium -> no rank:environmental samples 6 0.02% species:uncultured Nannocystaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Nannocystineae -> family:Nannocystaceae -> no rank:environmental samples 6 0.02% species:uncultured Geoalkalibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Desulfuromonadales -> family:Geobacteraceae -> genus:Geoalkalibacter -> no 6 0.02% 220 Finest Classification Taxonomy # % rank:environmental samples species:Arctic sea ice bacterium ARK10197 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Rhodobacteraceae -> genus:Octadecabacter -> no rank:unclassified Octadecabacter 5 0.01% species:Virgibacillus salexigens Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Bacillaceae -> genus:Virgibacillus 5 0.01% species:uncultured Syntrophorhabdaceae bacterium TA14 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Syntrophobacterales -> family:Syntrophorhabdaceae -> no rank:environmental samples 5 0.01% species:uncultured Nitriliruptor sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Nitriliruptoria -> order:Nitriliruptorales -> family:Nitriliruptoraceae -> genus:Nitriliruptor -> no rank:environmental samples 5 0.01% species:arsenite-oxidizing bacterium NT-5 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:unclassified Betaproteobacteria -> no rank:unclassified Betaproteobacteria (miscellaneous) 5 0.01% species:Streptomyces flavotricini Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Streptomycetales -> family:Streptomycetaceae -> genus:Streptomyces 5 0.01% species:uncultured Desulfomicrobium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Desulfovibrionales -> family:Desulfomicrobiaceae -> genus:Desulfomicrobium -> no rank:environmental samples 5 0.01% species:uncultured Rhodoferax sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Rhodoferax -> no rank:environmental samples 5 0.01% species:Bradyrhizobium sp. Kus-6 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Bradyrhizobiaceae -> genus:Bradyrhizobium 5 0.01% species:uncultured Endomicrobia bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Elusimicrobia -> class:Endomicrobia -> no rank:environmental samples 5 0.01% 221 Finest Classification Taxonomy # % species:Agrobacterium sp. BKBLPu7 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Rhizobiaceae -> no rank:Rhizobium/Agrobacterium group -> genus:Agrobacterium 5 0.01% species:uncultured Blastochloris sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Hyphomicrobiaceae -> genus:Blastochloris -> no rank:environmental samples 5 0.01% no rank:Limnothrix redekei NIVA-CYA 227/1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Cyanobacteria -> subclass:Oscillatoriophycideae -> order:Oscillatoriales -> genus:Limnothrix -> species:Limnothrix redekei 5 0.01% species:Variovorax sp. CH37-1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Variovorax 5 0.01% species:Microbacterium halophilum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Microbacteriaceae -> genus:Microbacterium 5 0.01% species:Dechlorobacter hydrogenophilus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Dechlorobacter 5 0.01% species:uncultured bacterium mle1-25 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Fibrobacteres/Acidobacteria group -> phylum:Acidobacteria -> class:Acidobacteriia -> order:Acidobacteriales -> no rank:environmental samples 5 0.01% species:bacterium enrichment culture clone B238(2011) Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 5 0.01% species:Paracoccus sp. TP-Snow-C3 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Rhodobacteraceae -> genus:Paracoccus 5 0.01% species:uncultured Mesorhizobium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Phyllobacteriaceae -> genus:Mesorhizobium -> no rank:environmental samples 5 0.01% no rank:Rhodospirillum photometricum DSM 122 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> family:Rhodospirillaceae -> genus:Pararhodospirillum -> species:Pararhodospirillum photometricum 5 0.01% species:uncultured Nitrosomonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Nitrosomonadales -> family:Nitrosomonadaceae -> genus:Nitrosomonas -> no rank:environmental samples 5 0.01% 222 Finest Classification Taxonomy # % species:beta proteobacterium Rufe28 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:unclassified Betaproteobacteria -> no rank:unclassified Betaproteobacteria (miscellaneous) 5 0.01% genus:Methylocystis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Methylocystaceae 5 0.01% species:uncultured Verrucomicrobiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobiaceae -> no rank:environmental samples 5 0.01% species:uncultured Parcubacteria bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> phylum:Parcubacteria -> no rank:environmental samples 5 0.01% species:uncultured Microgenomates bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> phylum:Microgenomates -> no rank:environmental samples 5 0.01% species:uncultured bacterium GC55 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Armatimonadetes -> no rank:environmental samples 5 0.01% species:uncultured cyanobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Cyanobacteria -> no rank:environmental samples 5 0.01% species:uncultured Desulfobacca sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Syntrophobacterales -> family:Syntrophaceae -> genus:Desulfobacca -> no rank:environmental samples 5 0.01% species:Castellaniella sp. 4.5A2 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Alcaligenaceae -> genus:Castellaniella 5 0.01% species:Chlorobaculum sp. BS3.2m-44-4full Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Chlorobi -> class:Chlorobia -> order:Chlorobiales -> family:Chlorobiaceae -> genus:Chlorobaculum 4 0.01% no rank:Senegalimassilia anaerobia JC110 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Coriobacteriia -> order:Coriobacteriales -> family:Coriobacteriaceae -> genus:Senegalimassilia -> species:Senegalimassilia anaerobia 4 0.01% species:uncultured Planctomyces sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> family:Planctomycetaceae -> genus:Planctomyces -> no rank:environmental samples 4 0.01% 223 Finest Classification Taxonomy # % species:uncultured bacterium tbr4-2 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:environmental samples 4 0.01% species:Clover phyllody phytoplasma Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Tenericutes -> class:Mollicutes -> order:Acholeplasmatales -> family:Acholeplasmataceae -> genus:Candidatus Phytoplasma -> species group:Candidatus Phytoplasma asteris 4 0.01% species:uncultured Sphingomonadaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Sphingomonadales -> family:Sphingomonadaceae -> no rank:environmental samples 4 0.01% species:Alicyclobacillus sp. TP7 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Alicyclobacillaceae -> genus:Alicyclobacillus 4 0.01% species:unidentified bacterium wb1_C17 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 4 0.01% species:uncultured Terrimonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Chitinophagaceae -> genus:Terrimonas -> no rank:environmental samples 4 0.01% species:uncultured Lachnospiraceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Lachnospiraceae -> no rank:environmental samples 4 0.01% species:uncultured Lysobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Lysobacter -> no rank:environmental samples 4 0.01% species:uncultured Chromatiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Chromatiales -> family:Chromatiaceae -> no rank:environmental samples 4 0.01% species:Idiomarina sp. G-He7 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Alteromonadales -> family:Idiomarinaceae -> genus:Idiomarina 4 0.01% species:Mesorhizobium sp. R38_Bhopal Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Phyllobacteriaceae -> genus:Mesorhizobium 4 0.01% species:uncultured Flavobacteriaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> order:Flavobacteriales -> family:Flavobacteriaceae -> no rank:environmental samples 4 0.01% 224 Finest Classification Taxonomy # % species:uncultured Synergistetes bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Synergistetes -> no rank:environmental samples 4 0.01% species:uncultured soil bacterium PBS-25 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 4 0.01% species:uncultured Haliangium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Nannocystineae -> family:Kofleriaceae -> genus:Haliangium -> no rank:environmental samples 4 0.01% species:Sphingomonas laterariae Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Sphingomonadales -> family:Sphingomonadaceae -> genus:Sphingomonas 4 0.01% species:Octadecabacter sp. IC146 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Rhodobacteraceae -> genus:Octadecabacter 4 0.01% species:Pseudomonas fluorescens Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Pseudomonadales -> family:Pseudomonadaceae -> genus:Pseudomonas -> species group:Pseudomonas fluorescens group 4 0.01% species:uncultured Nitrobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Bradyrhizobiaceae -> genus:Nitrobacter -> no rank:environmental samples 4 0.01% species:Edwardsiella tarda Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Enterobacteriales -> family:Enterobacteriaceae -> genus:Edwardsiella 4 0.01% species:Acinetobacter sp. ASR7 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Pseudomonadales -> family:Moraxellaceae -> genus:Acinetobacter 4 0.01% species:uncultured Hydrogenophilus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Hydrogenophilales -> family:Hydrogenophilaceae -> genus:Hydrogenophilus -> no rank:environmental samples 4 0.01% species:uncultured Caldilineales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Caldilineae -> order:Caldilineales -> no rank:environmental samples 4 0.01% species:Bacteroidia bacterium canine oral taxon 301 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> no rank:unclassified Bacteroidia 4 0.01% 225 Finest Classification Taxonomy # % species:uncultured Sterolibacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Sterolibacterium -> no rank:environmental samples 4 0.01% genus:Deinococcus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Deinococcus-Thermus -> class:Deinococci -> order:Deinococcales -> family:Deinococcaceae 4 0.01% genus:Vitreoscilla Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Neisseriales -> family:Neisseriaceae 4 0.01% species:uncultured Geobacillus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Bacillaceae -> genus:Geobacillus -> no rank:environmental samples 4 0.01% species:Chloroflexus aggregans Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Chloroflexia -> order:Chloroflexales -> suborder:Chloroflexineae -> family:Chloroflexaceae -> genus:Chloroflexus 4 0.01% species:bacterium enrichment culture clone B125(2011) Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 4 0.01% species:uncultured Methylococcaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Methylococcales -> family:Methylococcaceae -> no rank:environmental samples 3 0.01% species:Enterobacter sp. ICB113 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Enterobacteriales -> family:Enterobacteriaceae -> genus:Enterobacter 3 0.01% species:uncultured Conexibacteraceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Thermoleophilia -> order:Solirubrobacterales -> family:Conexibacteraceae -> no rank:environmental samples 3 0.01% species:Conexibacter sp. BS10 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Thermoleophilia -> order:Solirubrobacterales -> family:Conexibacteraceae -> genus:Conexibacter 3 0.01% species:uncultured Alicyclobacillus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Alicyclobacillaceae -> genus:Alicyclobacillus -> no rank:environmental samples 3 0.01% species:Streptomyces sp. SD-3 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Streptomycetales -> family:Streptomycetaceae -> genus:Streptomyces 3 0.01% species:uncultured Alcaligenaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Alcaligenaceae -> no rank:environmental samples 3 0.01% 226 Finest Classification Taxonomy # % species:Chryseobacterium sp. TB2-1-I Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> order:Flavobacteriales -> family:Flavobacteriaceae -> genus:Chryseobacterium 3 0.01% species:uncultured bacterium PHOS-HE51 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 3 0.01% species:Terrimonas sp. 16-45A Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Chitinophagaceae -> genus:Terrimonas 3 0.01% species:uncultured Marinilabiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Marinilabiliaceae -> no rank:environmental samples 3 0.01% species:Burkholderia sp. NAP1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Burkholderia 3 0.01% species:uncultured Banisveld landfill bacterium BVC47 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples -> no rank:Banisveld landfill bacteria ensemble 3 0.01% species:Arthrobacter sp. DK2009-3b Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Micrococcaceae -> genus:Arthrobacter 3 0.01% species:uncultured Synergistes sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Synergistetes -> class:Synergistia -> order:Synergistales -> family:Synergistaceae -> genus:Synergistes -> no rank:environmental samples 3 0.01% species:Virgibacillus halodenitrificans Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Bacillaceae -> genus:Virgibacillus 3 0.01% no rank:Mycoplasma mobile 163K Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Tenericutes -> class:Mollicutes -> order:Mycoplasmatales -> family:Mycoplasmataceae -> genus:Mycoplasma -> species:Mycoplasma mobile 3 0.01% species:uncultured Kofleriaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Nannocystineae -> family:Kofleriaceae -> no rank:environmental samples 3 0.01% 227 Finest Classification Taxonomy # % species:uncultured Runella sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Cytophagaceae -> genus:Runella -> no rank:environmental samples 3 0.01% species:Clostridiales bacterium canine oral taxon 157 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> no rank:unclassified Clostridiales -> no rank:unclassified Clostridiales (miscellaneous) 3 0.01% genus:Saprospira Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Saprospiraceae 3 0.01% species:Salinibacter sp. CH-10^6 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> order:Bacteroidetes Order II. Incertae sedis -> family:Rhodothermaceae -> genus:Salinibacter 3 0.01% species:Bifidobacterium indicum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Bifidobacteriales -> family:Bifidobacteriaceae -> genus:Bifidobacterium 3 0.01% species:Austwickia chelonae Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Dermatophilaceae -> genus:Austwickia 3 0.01% species:Chitinilyticum aquatile Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Neisseriales -> family:Chromobacteriaceae -> genus:Chitinilyticum 3 0.01% species:Brevibacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Brevibacteriaceae -> genus:Brevibacterium 3 0.01% species:Brevibacterium sp. K4-07D Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Brevibacteriaceae -> genus:Brevibacterium 3 0.01% species:Candidatus Glomeribacter gigasporarum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Candidatus Glomeribacter 3 0.01% species:Pandoraea sp. Y1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Pandoraea 3 0.01% species:Burkholderia fungorum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Burkholderia 3 0.01% 228 Finest Classification Taxonomy # % species:uncultured Oxalobacteraceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Oxalobacteraceae -> no rank:environmental samples 3 0.01% species:Nitrosomonas sp. NM 41 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Nitrosomonadales -> family:Nitrosomonadaceae -> genus:Nitrosomonas 3 0.01% species:Brevundimonas sp. BZ11 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Caulobacterales -> family:Caulobacteraceae -> genus:Brevundimonas 3 0.01% species:Streptomyces sp. CS Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Streptomycetales -> family:Streptomycetaceae -> genus:Streptomyces 3 0.01% species:Paracoccus sp. 'CJSPY1 (P-I)' Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Rhodobacteraceae -> genus:Paracoccus 3 0.01% species:uncultured Acidimicrobiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Acidimicrobiia -> order:Acidimicrobiales -> family:Acidimicrobiaceae -> no rank:environmental samples 3 0.01% species:Mesorhizobium sp. DLS-79 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Phyllobacteriaceae -> genus:Mesorhizobium 3 0.01% species:Xylella fastidiosa Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Xylella 3 0.01% species:Synergistales bacterium canine oral taxon 138 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Synergistetes -> class:Synergistia -> order:Synergistales -> no rank:unclassified Synergistales 3 0.01% species:Nostocoida limicola III Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:unclassified Planctomycetales 3 0.01% species:uncultured Nitrosospira sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Nitrosomonadales -> family:Nitrosomonadaceae -> genus:Nitrosospira -> no rank:environmental samples 3 0.01% species:uncultured Azospira sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Azospira -> no rank:environmental samples 3 0.01% no rank:unclassified Peptostreptococcaceae Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Peptostreptococcaceae 3 0.01% 229 Finest Classification Taxonomy # % species:Bdellovibrio sp. L Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Bdellovibrionales -> family:Bdellovibrionaceae -> genus:Bdellovibrio 3 0.01% species:uncultured Bacillus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Bacillaceae -> genus:Bacillus -> no rank:environmental samples 3 0.01% species:alpha proteobacterium GMD37D2 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:unclassified Alphaproteobacteria -> no rank:unclassified Alphaproteobacteria (miscellaneous) 3 0.01% species:Draconibacterium orientale Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Prolixibacteraceae -> genus:Draconibacterium 3 0.01% species:Emticicia sp. JJ014 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Cytophagaceae -> genus:Emticicia 3 0.01% species:uncultured Salinibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> order:Bacteroidetes Order II. Incertae sedis -> family:Rhodothermaceae -> genus:Salinibacter -> no rank:environmental samples 3 0.01% species:uncultured sludge bacterium S14 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> no rank:environmental samples 3 0.01% species:marine bacterium NAMAF003 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 3 0.01% species:uncultured Treponema sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Spirochaetes -> class:Spirochaetia -> order:Spirochaetales -> family:Spirochaetaceae -> genus:Treponema -> no rank:environmental samples 3 0.01% species:bacterium enrichment culture clone ecb8 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 3 0.01% species:uncultured Arcobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Epsilonproteobacteria -> order:Campylobacterales -> family:Campylobacteraceae -> genus:Arcobacter -> no rank:environmental samples 3 0.01% 230 Finest Classification Taxonomy # % species:Megasphaera elsdenii Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Negativicutes -> order:Selenomonadales -> family:Veillonellaceae -> genus:Megasphaera 2 0.01% genus:Niabella Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Chitinophagaceae 2 0.01% species:Brevibacillus sp. YS-2 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Paenibacillaceae -> genus:Brevibacillus 2 0.01% species:Denitratisoma oestradiolicum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Denitratisoma 2 0.01% species:uncultured bacterium H25 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> no rank:environmental samples 2 0.01% species:Azoarcus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Azoarcus 2 0.01% species:bacterium enrichment culture clone R4-52B Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 2 0.01% species:Corynebacterium tuberculostearicum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Corynebacteriales -> family:Corynebacteriaceae -> genus:Corynebacterium 2 0.01% species:Comamonadaceae bacterium oral taxon F47 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> no rank:unclassified Comamonadaceae 2 0.01% species:Hydrogenophaga sp. PBC Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Hydrogenophaga 2 0.01% species:Variovorax paradoxus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Variovorax 2 0.01% species:Acidovorax wohlfahrtii Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Acidovorax 2 0.01% species:Oxalobacter formigenes Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Oxalobacteraceae -> genus:Oxalobacter 2 0.01% 231 Finest Classification Taxonomy # % species:Advenella kashmirensis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Alcaligenaceae -> genus:Advenella 2 0.01% species:uncultured Propionibacteriaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Propionibacteriales -> family:Propionibacteriaceae -> no rank:environmental samples 2 0.01% species:Phenylobacterium immobile Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Caulobacterales -> family:Caulobacteraceae -> genus:Phenylobacterium 2 0.01% species:Cereibacter changlensis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Rhodobacteraceae -> genus:Cereibacter 2 0.01% species:Rhodovulum sulfidophilum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Rhodobacteraceae -> genus:Rhodovulum 2 0.01% species:Azospirillum lipoferum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> family:Rhodospirillaceae -> genus:Azospirillum 2 0.01% species:uncultured Acetobacteraceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> family:Acetobacteraceae -> no rank:environmental samples 2 0.01% species:Nonomuraea spiralis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Streptosporangiales -> family:Streptosporangiaceae -> genus:Nonomuraea 2 0.01% species:bacterium Ellin5134 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> family:Acetobacteraceae -> no rank:unclassified Acetobacteraceae 2 0.01% species:Nocardiopsis sp. AE3 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Streptosporangiales -> family:Nocardiopsaceae -> genus:Nocardiopsis 2 0.01% species:Frankia sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Frankiales -> family:Frankiaceae -> genus:Frankia 2 0.01% species:Mesorhizobium sp. AC100e Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Phyllobacteriaceae -> genus:Mesorhizobium 2 0.01% species:Candidatus Anammoxoglobus propionicus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Candidatus Brocadiales -> family:Candidatus Brocadiaceae -> genus:Candidatus 2 0.01% 232 Finest Classification Taxonomy # % Anammoxoglobus species:Acinetobacter sp. enrichment culture clone PH108 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Pseudomonadales -> family:Moraxellaceae -> genus:Acinetobacter -> no rank:environmental samples 2 0.01% species:Moraxella sp. canine oral taxon 017 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Pseudomonadales -> family:Moraxellaceae -> genus:Moraxella -> subgenus:Moraxella 2 0.01% species:Bradyrhizobium japonicum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Bradyrhizobiaceae -> genus:Bradyrhizobium 2 0.01% species:Bradyrhizobium sp. vgn-1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Bradyrhizobiaceae -> genus:Bradyrhizobium 2 0.01% species:Bradyrhizobium sp. JR022 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Bradyrhizobiaceae -> genus:Bradyrhizobium 2 0.01% species:uncultured Thioalkalispira sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Chromatiales -> family:Thioalkalispiraceae -> genus:Thioalkalispira -> no rank:environmental samples 2 0.01% species:Methylosinus sp. 24-21 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Methylocystaceae -> genus:Methylosinus 2 0.01% no rank:Frateuria aurantia DSM 6220 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Frateuria -> species:Frateuria aurantia 2 0.01% species:uncultured Shewanella sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Alteromonadales -> family:Shewanellaceae -> genus:Shewanella -> no rank:environmental samples 2 0.01% species:Pseudoalteromonas sp. BSw20410 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Alteromonadales -> family:Pseudoalteromonadaceae -> genus:Pseudoalteromonas 2 0.01% species:Halomonas campisalis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Oceanospirillales -> family:Halomonadaceae -> genus:Halomonas 2 0.01% 233 Finest Classification Taxonomy # % species:Escherichia coli Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Enterobacteriales -> family:Enterobacteriaceae -> genus:Escherichia 2 0.01% species:Psychrobacter sp. 7321 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Pseudomonadales -> family:Moraxellaceae -> genus:Psychrobacter 2 0.01% species:uncultured Sphingobium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Sphingomonadales -> family:Sphingomonadaceae -> genus:Sphingobium -> no rank:environmental samples 2 0.01% species:uncultured Magnetococcus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Magnetococcales -> family:Magnetococcaceae -> genus:Magnetococcus -> no rank:environmental samples 2 0.01% species:uncultured Polaromonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Polaromonas -> no rank:environmental samples 2 0.01% species:Tepidimonas thermarum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> no rank:unclassified Burkholderiales -> no rank:Burkholderiales Genera incertae sedis -> genus:Tepidimonas 2 0.01% species:Chromobacterium sp. 44 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Neisseriales -> family:Chromobacteriaceae -> no rank:Chromobacterium group -> genus:Chromobacterium 2 0.01% genus:Nocardioides Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Propionibacteriales -> family:Nocardioidaceae 2 0.01% genus:Leucobacter Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Microbacteriaceae 2 0.01% genus:Ornithinicoccus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Intrasporangiaceae 2 0.01% species:Boyliae praeputiale Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> no rank:unclassified Actinobacteria -> genus:Boyliae 2 0.01% species:Bacteroidetes bacterium NeomS2D4 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:unclassified Bacteroidetes -> no rank:unclassified Bacteroidetes (miscellaneous) 2 0.01% 234 Finest Classification Taxonomy # % species:uncultured Spartobacteria bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Spartobacteria -> no rank:environmental samples 2 0.01% species:Desulfovibrio burkinensis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Desulfovibrionales -> family:Desulfovibrionaceae -> genus:Desulfovibrio 2 0.01% species:sulfate-reducing bacterium TRM1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Desulfobacterales -> family:Desulfobulbaceae -> no rank:unclassified Desulfobulbaceae 2 0.01% species:Wolinella succinogenes Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Epsilonproteobacteria -> order:Campylobacterales -> family:Helicobacteraceae -> genus:Wolinella 2 0.01% genus:Aquaspirillum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Neisseriales -> family:Chromobacteriaceae 2 0.01% species:beta proteobacterium JN18_V17_A3 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:unclassified Betaproteobacteria -> no rank:unclassified Betaproteobacteria (miscellaneous) 2 0.01% species:Desulfosporosinus sp. enrichment culture clone Dd Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Peptococcaceae -> genus:Desulfosporosinus -> no rank:environmental samples 2 0.01% species:uncultured Leptotrichia sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Fusobacteria -> class:Fusobacteriia -> order:Fusobacteriales -> family:Leptotrichiaceae -> genus:Leptotrichia -> no rank:environmental samples 2 0.01% no rank:Sebaldella termitidis ATCC 33386 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Fusobacteria -> class:Fusobacteriia -> order:Fusobacteriales -> family:Leptotrichiaceae -> genus:Sebaldella -> species:Sebaldella termitidis 2 0.01% species:Lyngbya sp. BAN TS02 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Cyanobacteria -> subclass:Oscillatoriophycideae -> order:Oscillatoriales -> genus:Lyngbya 2 0.01% species:Verrucomicrobiaceae bacterium CHC12 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobiaceae -> 2 0.01% 235 Finest Classification Taxonomy # % no rank:unclassified Verrucomicrobiaceae species:Pelagicoccus croceus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Opitutae -> order:Puniceicoccales -> family:Puniceicoccaceae -> genus:Pelagicoccus 2 0.01% species:Candidatus Protochlamydia sp. cvE12 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Chlamydiae -> class:Chlamydiia -> order:Chlamydiales -> family:Parachlamydiaceae -> genus:Candidatus Protochlamydia 2 0.01% species:Pedobacter sp. BAL39 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Sphingobacteriaceae -> genus:Pedobacter 2 0.01% species:Lewinella persica Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Saprospiraceae -> genus:Lewinella 2 0.01% species:Anaerocella delicata Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Rikenellaceae -> genus:Anaerocella 2 0.01% species:uncultured verrucomicrobium DEV003 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> no rank:environmental samples 2 0.01% species:uncultured candidate division JS1 bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:candidate division JS1 -> no rank:environmental samples 2 0.01% species:uncultured Caldithrix sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> genus:Caldithrix -> no rank:environmental samples 2 0.01% species:Flexibacter roseolus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Cytophagaceae -> genus:Flexibacter 2 0.01% species:uncultured Gram-positive bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Gram-positive bacteria -> no rank:environmental samples 2 0.01% 236 Finest Classification Taxonomy # % species:Sediminitomix flava Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Flammeovirgaceae -> genus:Sediminitomix 2 0.01% species:uncultured Pietermaritzburg bacterium Y17-2 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 2 0.01% species:uncultured Gemmatimonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Gemmatimonadetes -> class:Gemmatimonadetes -> order:Gemmatimonadales -> family:Gemmatimonadaceae -> genus:Gemmatimonas -> no rank:environmental samples 2 0.01% species:uncultured sludge bacterium A3 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 2 0.01% species:uncultured Thermomicrobium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Thermomicrobia -> order:Thermomicrobiales -> family:Thermomicrobiaceae -> genus:Thermomicrobium -> no rank:environmental samples 2 0.01% species:uncultured Anaerolinea sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Anaerolineae -> order:Anaerolineales -> family:Anaerolineaceae -> genus:Anaerolinea -> no rank:environmental samples 2 0.01% species:uncultured Dehalococcoides sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Dehalococcoidia -> order:Dehalococcoidales -> family:Dehalococcoidaceae -> genus:Dehalococcoides -> no rank:environmental samples 2 0.01% species:uncultured Actinomyces sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Actinomycetales -> family:Actinomycetaceae -> genus:Actinomyces -> no rank:environmental samples 2 0.01% species:uncultured Brachybacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Dermabacteraceae -> genus:Brachybacterium -> no rank:environmental samples 2 0.01% no rank:Gryllotalpicola ginsengisoli DSM 22003 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Microbacteriaceae -> genus:Gryllotalpicola -> species:Gryllotalpicola ginsengisoli 2 0.01% species:groundwater planktonic bacterium Z1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 2 0.01% species:bacterium AEOC021 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 2 0.01% 237 Finest Classification Taxonomy # % species:uncultured Geobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Desulfuromonadales -> family:Geobacteraceae -> genus:Geobacter -> no rank:environmental samples 2 0.01% species:uncultured Desulfovibrio sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Desulfovibrionales -> family:Desulfovibrionaceae -> genus:Desulfovibrio -> no rank:environmental samples 2 0.01% species:uncultured bacterium SHA-28 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 2 0.01% species:uncultured Barnesiella sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Porphyromonadaceae -> genus:Barnesiella -> no rank:environmental samples 2 0.01% species:uncultured Prevotella sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Prevotellaceae -> genus:Prevotella -> no rank:environmental samples 2 0.01% species:uncultured Ignavibacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Ignavibacteriae -> class:Ignavibacteria -> order:Ignavibacteriales -> family:Ignavibacteriaceae -> genus:Ignavibacterium -> no rank:environmental samples 2 0.01% species:bacterium enrichment culture clone DPF33 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 2 0.01% others   1253 3.65% Total  34356 100.00%    238 Appendix C  The identified species and their percentages of the samples from the ozonated  SBR Finest Classification Taxonomy # % species:uncultured planctomycete Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:environmental samples 4821 11.91% species:uncultured beta proteobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:environmental samples 4343 10.73% species:uncultured Gemmatimonadetes bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Gemmatimonadetes -> no rank:environmental samples 4203 10.38% species:uncultured soil bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 3587 8.86% species:uncultured alpha proteobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 2781 6.87% species:uncultured Bacteroidetes bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 2673 6.60% species:uncultured sludge bacterium S36 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 2472 6.11% species:uncultured gamma proteobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> no rank:environmental samples 1528 3.77% species:uncultured delta proteobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> no rank:environmental samples 1331 3.29% species:uncultured sludge bacterium H2 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> no rank:environmental samples 838 2.07% species:uncultured Verrucomicrobia bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> no rank:environmental samples 452 1.12% species:uncultured Bacterial: no rank:cellular organisms -> 440 1.09% 239 Finest Classification Taxonomy # % Acidobacteria bacterium superkingdom:Bacteria -> superphylum:Fibrobacteres/Acidobacteria group -> phylum:Acidobacteria -> no rank:environmental samples species:uncultured Stigmatella sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Cystobacterineae -> family:Cystobacteraceae -> genus:Stigmatella -> no rank:environmental samples 422 1.04% species:uncultured Planctomycetales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:environmental samples 354 0.87% species:uncultured Candidatus Accumulibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:unclassified Betaproteobacteria -> genus:Candidatus Accumulibacter -> no rank:environmental samples 335 0.83% species:uncultured Chloroflexi bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> no rank:environmental samples 331 0.82% species:uncultured actinobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> no rank:environmental samples 331 0.82% species:uncultured sludge bacterium A17 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:environmental samples 289 0.71% species:uncultured Gemmatimonadaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Gemmatimonadetes -> class:Gemmatimonadetes -> order:Gemmatimonadales -> family:Gemmatimonadaceae -> no rank:environmental samples 252 0.62% species:Prosthecobacter vanneervenii Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobiaceae -> genus:Prosthecobacter 243 0.60% species:uncultured Comamonadaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> no rank:environmental samples 214 0.53% species:uncultured Sphingobacteriales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> no rank:environmental samples 191 0.47% species:uncultured proteobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> no 189 0.47% 240 Finest Classification Taxonomy # % rank:environmental samples species:uncultured Intrasporangiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Intrasporangiaceae -> no rank:environmental samples 187 0.46% species:uncultured Bacteroides sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Bacteroidaceae -> genus:Bacteroides -> no rank:environmental samples 171 0.42% species:uncultured Rhodospirillales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> no rank:environmental samples 165 0.41% species:uncultured sludge bacterium A27b Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 157 0.39% species:Brevifollis gellanilyticus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobiaceae -> genus:Brevifollis 156 0.39% species:uncultured Dechloromonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Dechloromonas -> no rank:environmental samples 140 0.35% species:uncultured Xanthomonadaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> no rank:environmental samples 135 0.33% species:Flavobacterium sp. HWG-A1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> order:Flavobacteriales -> family:Flavobacteriaceae -> genus:Flavobacterium 129 0.32% species:Arenimonas aquatica Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Arenimonas 124 0.31% species:Prosthecobacter fluviatilis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobiaceae -> genus:Prosthecobacter 123 0.30% species:uncultured rumen bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental 120 0.30% 241 Finest Classification Taxonomy # % samples species:Curvibacter putative symbiont of Hydra magnipapillata Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Curvibacter 115 0.28% species:uncultured Rhodocyclaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> no rank:environmental samples 104 0.26% species:uncultured Chitinophagaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Chitinophagaceae -> no rank:environmental samples 99 0.24% species:uncultured Gemmata sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> family:Planctomycetaceae -> genus:Gemmata -> no rank:environmental samples 98 0.24% species:uncultured Burkholderiales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> no rank:environmental samples 92 0.23% species:uncultured Termite group 1 bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Elusimicrobia -> no rank:environmental samples 92 0.23% species:uncultured Gram-positive bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Gram-positive bacteria -> no rank:environmental samples 86 0.21% species:uncultured Verminephrobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Verminephrobacter -> no rank:environmental samples 85 0.21% species:uncultured Achromobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Alcaligenaceae -> genus:Achromobacter -> no rank:environmental samples 83 0.20% species:Woodsholea maritima Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Caulobacterales -> family:Caulobacteraceae -> genus:Woodsholea 83 0.20% species:Comamonas denitrificans Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Comamonas 82 0.20% species:uncultured Planctomycetaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> family:Planctomycetaceae -> no rank:environmental 82 0.20% 242 Finest Classification Taxonomy # % samples species:bacterium GPB6 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 82 0.20% species:uncultured deep-sea bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 79 0.20% species:uncultured Chlorobi bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Chlorobi -> no rank:environmental samples 74 0.18% species:Dokdonella immobilis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Dokdonella 74 0.18% species:uncultured marine bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 71 0.18% species:Candidatus Halomonas phosphatis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Oceanospirillales -> family:Halomonadaceae -> genus:Halomonas 69 0.17% species:uncultured bacterium PHOS-HE21 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 69 0.17% species:uncultured Rhizobiales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> no rank:environmental samples 66 0.16% species:uncultured Bacteroidales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> no rank:environmental samples 64 0.16% species:Austwickia chelonae Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Dermatophilaceae -> genus:Austwickia 64 0.16% species:Brachymonas sp. canine oral taxon 015 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Brachymonas 58 0.14% species:uncultured sludge bacterium H8 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:environmental samples 53 0.13% species:uncultured Nitrospira sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Nitrospirae -> class:Nitrospira -> order:Nitrospirales -> family:Nitrospiraceae -> genus:Nitrospira -> no rank:environmental samples 50 0.12% species:uncultured Brevundimonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> 49 0.12% 243 Finest Classification Taxonomy # % class:Alphaproteobacteria -> order:Caulobacterales -> family:Caulobacteraceae -> genus:Brevundimonas -> no rank:environmental samples genus:Cellvibrio Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Cellvibrionales -> family:Cellvibrionaceae 49 0.12% genus:Uliginosibacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae 47 0.12% species:Flavobacterium sp. HME6144 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> order:Flavobacteriales -> family:Flavobacteriaceae -> genus:Flavobacterium 44 0.11% species:uncultured Acetanaerobacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Ruminococcaceae -> genus:Acetanaerobacterium -> no rank:environmental samples 42 0.10% species:uncultured Nannocystaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Nannocystineae -> family:Nannocystaceae -> no rank:environmental samples 41 0.10% species:uncultured Gallionella sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Gallionellales -> family:Gallionellaceae -> genus:Gallionella -> no rank:environmental samples 38 0.09% species:uncultured Firmicutes bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> no rank:environmental samples 38 0.09% species:Pyrinomonas methylaliphatogenes Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Fibrobacteres/Acidobacteria group -> phylum:Acidobacteria -> no rank:unclassified Acidobacteria -> no rank:Acidobacteria subdivision 4 -> genus:Pyrinomonas 36 0.09% species:uncultured Crater Lake bacterium CL500-15 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples -> no rank:Crater Lake bacteria ensemble 36 0.09% species:uncultured compost bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 36 0.09% species:uncultured Sphingobacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Sphingobacteriaceae 36 0.09% 244 Finest Classification Taxonomy # % -> genus:Sphingobacterium -> no rank:environmental samples species:uncultured Burkholderia sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Burkholderia -> no rank:environmental samples 34 0.08% species:Nitrobacter winogradskyi Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Bradyrhizobiaceae -> genus:Nitrobacter 33 0.08% species:uncultured Hyphomicrobiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Hyphomicrobiaceae -> no rank:environmental samples 32 0.08% species:uncultured Flavobacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> order:Flavobacteriales -> family:Flavobacteriaceae -> genus:Flavobacterium -> no rank:environmental samples 30 0.07% species:uncultured Armatimonadetes bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Armatimonadetes -> no rank:environmental samples 29 0.07% species:halophilic eubacterium EHB-4 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> order:Bacteroidetes Order II. Incertae sedis -> family:Rhodothermaceae -> genus:Salinibacter -> no rank:unclassified Salinibacter 28 0.07% species:Rhodoferax sp. IMCC1723 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Rhodoferax 28 0.07% species:uncultured Acinetobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Pseudomonadales -> family:Moraxellaceae -> genus:Acinetobacter -> no rank:environmental samples 26 0.06% species:uncultured Clostridiales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> no rank:environmental samples 26 0.06% species:Paracraurococcus sp. ORS 1473 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> family:Acetobacteraceae -> genus:Paracraurococcus 26 0.06% species:uncultured halophilic eubacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 25 0.06% 245 Finest Classification Taxonomy # % species:uncultured Gemmatimonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Gemmatimonadetes -> class:Gemmatimonadetes -> order:Gemmatimonadales -> family:Gemmatimonadaceae -> genus:Gemmatimonas -> no rank:environmental samples 25 0.06% species:Blastochloris viridis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Hyphomicrobiaceae -> genus:Blastochloris 25 0.06% species:uncultured bacterium PHOS-HE28 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 25 0.06% species:Xanthomonas sp. enrichment culture clone VanCtr94 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Xanthomonas -> no rank:environmental samples 24 0.06% species:uncultured Dysgonomonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Porphyromonadaceae -> genus:Dysgonomonas -> no rank:environmental samples 24 0.06% species:Bacillus subtilis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Bacillaceae -> genus:Bacillus -> species group:Bacillus subtilis group 23 0.06% species:Nitrospira sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Nitrospirae -> class:Nitrospira -> order:Nitrospirales -> family:Nitrospiraceae -> genus:Nitrospira 23 0.06% species:uncultured Endomicrobia bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Elusimicrobia -> class:Endomicrobia -> no rank:environmental samples 22 0.05% species:uncultured Bradyrhizobium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Bradyrhizobiaceae -> genus:Bradyrhizobium -> no rank:environmental samples 22 0.05% species:uncultured Thiobacillus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Hydrogenophilales -> family:Hydrogenophilaceae -> genus:Thiobacillus -> no rank:environmental samples 22 0.05% species:uncultured sludge bacterium A41 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 22 0.05% species:Pelomonas Bacterial: no rank:cellular organisms -> 21 0.05% 246 Finest Classification Taxonomy # % puraquae superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Pelomonas species:uncultured Sphingobacteria bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> no rank:environmental samples 20 0.05% species:uncultured Deferribacteres bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Deferribacteres -> class:Deferribacteres -> no rank:environmental samples 20 0.05% species:uncultured Hyphomicrobium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Hyphomicrobiaceae -> genus:Hyphomicrobium -> no rank:environmental samples 20 0.05% species:uncultured sludge bacterium S9 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> no rank:environmental samples 19 0.05% species:Arenimonas sp. PYM3-14 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Arenimonas 18 0.04% species:Caulobacter sp. H62 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Caulobacterales -> family:Caulobacteraceae -> genus:Caulobacter 18 0.04% species:uncultured cyanobacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Cyanobacteria -> no rank:environmental samples 18 0.04% no rank:environmental samples Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Fusobacteria -> class:Fusobacteriia -> order:Fusobacteriales -> family:Leptotrichiaceae -> genus:Leptotrichia 18 0.04% species:uncultured bacterium VC2.1 Bac16 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 17 0.04% species:Acholeplasma cavigenitalium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Tenericutes -> class:Mollicutes -> order:Acholeplasmatales -> family:Acholeplasmataceae -> genus:Acholeplasma 17 0.04% species:beta proteobacterium pACH94 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:unclassified Betaproteobacteria -> no rank:unclassified Betaproteobacteria (miscellaneous) 17 0.04% species:uncultured Nitrosomonadaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Nitrosomonadales -> family:Nitrosomonadaceae -> no rank:environmental samples 16 0.04% species:uncultured Arcobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> 16 0.04% 247 Finest Classification Taxonomy # % class:Epsilonproteobacteria -> order:Campylobacterales -> family:Campylobacteraceae -> genus:Arcobacter -> no rank:environmental samples species:uncultured Halanaerobiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Halanaerobiales -> family:Halanaerobiaceae -> no rank:environmental samples 15 0.04% species:alpha proteobacterium A0839 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:unclassified Alphaproteobacteria -> no rank:unclassified Alphaproteobacteria (miscellaneous) 15 0.04% species:Thiobacillus Q Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Hydrogenophilales -> family:Hydrogenophilaceae -> genus:Thiobacillus 15 0.04% species:uncultured Flavobacteriia bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> no rank:environmental samples 15 0.04% no rank:Paludibacter propionicigenes WB4 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Porphyromonadaceae -> genus:Paludibacter -> species:Paludibacter propionicigenes 15 0.04% species:Crenothrix polyspora Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Methylococcales -> family:Crenotrichaceae -> genus:Crenothrix 15 0.04% species:Delftia sp. RF-93 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Delftia 14 0.03% species:uncultured Comamonas sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Comamonas -> no rank:environmental samples 14 0.03% species:uncultured Acidobacteriaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Fibrobacteres/Acidobacteria group -> phylum:Acidobacteria -> class:Acidobacteriia -> order:Acidobacteriales -> family:Acidobacteriaceae -> no rank:environmental samples 14 0.03% species:Ensifer adhaerens Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Rhizobiaceae -> no rank:Sinorhizobium/Ensifer group -> genus:Ensifer 14 0.03% species:Stella humosa Bacterial: no rank:cellular organisms -> 14 0.03% 248 Finest Classification Taxonomy # % superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> family:Acetobacteraceae -> genus:Stella no rank:Gemmatimonas aurantiaca T-27 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Gemmatimonadetes -> class:Gemmatimonadetes -> order:Gemmatimonadales -> family:Gemmatimonadaceae -> genus:Gemmatimonas -> species:Gemmatimonas aurantiaca 14 0.03% genus:Nakamurella Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Nakamurellales -> family:Nakamurellaceae 14 0.03% species:uncultured Haliangium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Nannocystineae -> family:Kofleriaceae -> genus:Haliangium -> no rank:environmental samples 14 0.03% species:uncultured sludge bacterium A7 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 13 0.03% species:Flavobacterium sp. JJ004 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> order:Flavobacteriales -> family:Flavobacteriaceae -> genus:Flavobacterium 13 0.03% species:uncultured Epsilonproteobacteria bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Epsilonproteobacteria -> no rank:environmental samples 13 0.03% species:Acidovorax defluvii Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Acidovorax 13 0.03% species:uncultured Acidimicrobiales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Acidimicrobiia -> order:Acidimicrobiales -> no rank:environmental samples 13 0.03% species:beta proteobacterium Rufe28 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:unclassified Betaproteobacteria -> no rank:unclassified Betaproteobacteria (miscellaneous) 12 0.03% species:uncultured sediment bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 12 0.03% species:Nitrosomonas sp. Nm59 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Nitrosomonadales -> family:Nitrosomonadaceae -> genus:Nitrosomonas 12 0.03% 249 Finest Classification Taxonomy # % species:uncultured bacterium PHOS-HE25 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 12 0.03% species:Phormidium autumnale Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Cyanobacteria -> subclass:Oscillatoriophycideae -> order:Oscillatoriales -> genus:Phormidium 12 0.03% species:agricultural soil bacterium SC-I-12 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples -> no rank:agricultural soil bacteria ensemble 12 0.03% species:Laribacter hongkongensis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Neisseriales -> family:Chromobacteriaceae -> genus:Laribacter 12 0.03% species:Sphingomonas sp. BAC151 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Sphingomonadales -> family:Sphingomonadaceae -> genus:Sphingomonas 12 0.03% species:uncultured Gemmatimonadales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Gemmatimonadetes -> class:Gemmatimonadetes -> order:Gemmatimonadales -> no rank:environmental samples 11 0.03% species:uncultured Ruminococcaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Ruminococcaceae -> no rank:environmental samples 11 0.03% species:gamma proteobacterium HdN1 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> no rank:unclassified Gammaproteobacteria -> no rank:unclassified Gammaproteobacteria (miscellaneous) 11 0.03% species:uncultured Caulobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Caulobacterales -> family:Caulobacteraceae -> genus:Caulobacter -> no rank:environmental samples 11 0.03% species:Hyphomicrobium sp. Ellin112 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Hyphomicrobiaceae -> genus:Hyphomicrobium 11 0.03% species:uncultured sludge bacterium S14 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> no rank:environmental samples 11 0.03% species:Micromonospora sp. 215008 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micromonosporales -> family:Micromonosporaceae -> genus:Micromonospora 11 0.03% species:bacterium WHC2-6 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:unclassified Bacteria -> no rank:unclassified Bacteria (miscellaneous) 11 0.03% 250 Finest Classification Taxonomy # % species:uncultured Denitratisoma sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Denitratisoma -> no rank:environmental samples 11 0.03% species:uncultured Veillonellaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Negativicutes -> order:Selenomonadales -> family:Veillonellaceae -> no rank:environmental samples 11 0.03% species:uncultured Phycisphaerales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Phycisphaerae -> order:Phycisphaerales -> no rank:environmental samples 11 0.03% species:Brevundimonas sp. BZ11 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Caulobacterales -> family:Caulobacteraceae -> genus:Brevundimonas 10 0.02% species:arsenite-oxidizing bacterium NT-5 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:unclassified Betaproteobacteria -> no rank:unclassified Betaproteobacteria (miscellaneous) 10 0.02% species:uncultured Cystobacterineae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Myxococcales -> suborder:Cystobacterineae -> no rank:environmental samples 10 0.02% species:uncultured Bdellovibrionales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Bdellovibrionales -> no rank:environmental samples 10 0.02% species:uncultured Brochothrix sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Listeriaceae -> genus:Brochothrix -> no rank:environmental samples 10 0.02% species:uncultured Rubrobacteria bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Rubrobacteria -> no rank:environmental samples 10 0.02% species:uncultured Hydrogenophaga sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Hydrogenophaga -> no rank:environmental samples 10 0.02% species:uncultured Ktedonobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Ktedonobacteria -> order:Ktedonobacterales -> family:Ktedonobacteraceae -> genus:Ktedonobacter -> no rank:environmental samples 10 0.02% species:[Brevibacterium] halotolerans Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Bacillaceae -10 0.02% 251 Finest Classification Taxonomy # % > genus:Bacillus genus:Cesiribacter Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Flammeovirgaceae 10 0.02% species:Lacibacter sp. JJ009 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Chitinophagaceae -> genus:Lacibacter 10 0.02% no rank:Thioclava pacifica DSM 10166 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Rhodobacteraceae -> genus:Thioclava -> species:Thioclava pacifica 10 0.02% species:uncultured Curvibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Curvibacter -> no rank:environmental samples 10 0.02% species:uncultured Desulfobacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Desulfobacterales -> family:Desulfobacteraceae -> genus:Desulfobacterium -> no rank:environmental samples 9 0.02% species:Enterobacter sp. ICB113 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Enterobacteriales -> family:Enterobacteriaceae -> genus:Enterobacter 9 0.02% species:Cytophaga hutchinsonii Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Cytophagaceae -> genus:Cytophaga 9 0.02% species:uncultured Nitrospirae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Nitrospirae -> no rank:environmental samples 9 0.02% species:Shewanella sp. HJ-53 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Alteromonadales -> family:Shewanellaceae -> genus:Shewanella 9 0.02% species:uncultured Desulfuromonadales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Desulfuromonadales -> no rank:environmental samples 9 0.02% species:uncultured Eubacteriaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Eubacteriaceae -> no rank:environmental 9 0.02% 252 Finest Classification Taxonomy # % samples species:bacterium enrichment culture clone R4-80B Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 9 0.02% species:Pseudofulvimonas gallinarii Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Xanthomonadales -> family:Xanthomonadaceae -> genus:Pseudofulvimonas 9 0.02% species:uncultured Verrucomicrobiales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> no rank:environmental samples 9 0.02% species:uncultured Planctomyces sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> family:Planctomycetaceae -> genus:Planctomyces -> no rank:environmental samples 9 0.02% species:uncultured Caldilineaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Caldilineae -> order:Caldilineales -> family:Caldilineaceae -> no rank:environmental samples 9 0.02% species:Bacteroides nordii Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Bacteroidaceae -> genus:Bacteroides 9 0.02% species:uncultured Cloacibacillus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Synergistetes -> class:Synergistia -> order:Synergistales -> family:Synergistaceae -> genus:Cloacibacillus -> no rank:environmental samples 9 0.02% species:uncultured sludge bacterium A26 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> no rank:environmental samples 9 0.02% species:uncultured Caldilinea sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Chloroflexi -> class:Caldilineae -> order:Caldilineales -> family:Caldilineaceae -> genus:Caldilinea -> no rank:environmental samples 9 0.02% species:Methylosinus sp. 24-21 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Methylocystaceae -> genus:Methylosinus 9 0.02% species:uncultured Paludibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Bacteroidia -> order:Bacteroidales -> family:Porphyromonadaceae -> 9 0.02% 253 Finest Classification Taxonomy # % genus:Paludibacter -> no rank:environmental samples species:uncultured Alicyclobacillus sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Alicyclobacillaceae -> genus:Alicyclobacillus -> no rank:environmental samples 8 0.02% species:uncultured bacterium MS8 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> no rank:environmental samples 8 0.02% species:Phycicoccus jejuensis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Intrasporangiaceae -> genus:Phycicoccus 8 0.02% species:uncultured forest soil bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 8 0.02% species:uncultured low G+C Gram-positive bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> no rank:environmental samples 8 0.02% species:unidentified Verrucomicrobium group OPB35 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobia subdivision 3 -> no rank:environmental samples 8 0.02% species:uncultured Haliscomenobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Sphingobacteriia -> order:Sphingobacteriales -> family:Saprospiraceae -> genus:Haliscomenobacter -> no rank:environmental samples 8 0.02% species:unidentified bacterium wb1_C17 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 8 0.02% species:Achromobacter xylosoxidans Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Alcaligenaceae -> genus:Achromobacter 8 0.02% species:Herbaspirillum sp. TSO33-2 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Oxalobacteraceae -> genus:Herbaspirillum 8 0.02% species:uncultured ammonia-oxidizing bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples -> no rank:ammonia oxidising bacteria ensemble 8 0.02% species:uncultured Flavobacteriaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Flavobacteriia -> order:Flavobacteriales -> family:Flavobacteriaceae -> 8 0.02% 254 Finest Classification Taxonomy # % no rank:environmental samples species:uncultured Acidovorax sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Acidovorax -> no rank:environmental samples 8 0.02% genus:Ornithinicoccus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Intrasporangiaceae 8 0.02% genus:Vitreoscilla Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Neisseriales -> family:Neisseriaceae 8 0.02% species:uncultured bacterium MK09 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:environmental samples 8 0.02% species:uncultured endolithic bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 8 0.02% species:Agrobacterium sp. BKBLPu7 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Rhizobiaceae -> no rank:Rhizobium/Agrobacterium group -> genus:Agrobacterium 7 0.02% species:Candidatus Glomeribacter gigasporarum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Burkholderiaceae -> genus:Candidatus Glomeribacter 7 0.02% species:Paenibacillus sp. XWS-29 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> order:Bacillales -> family:Paenibacillaceae -> genus:Paenibacillus 7 0.02% species:uncultured Alcaligenaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Alcaligenaceae -> no rank:environmental samples 7 0.02% species:uncultured Rubrobacterales bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Rubrobacteria -> order:Rubrobacterales -> no rank:environmental samples 7 0.02% species:Planctomycetales bacterium Ellin7244 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Planctomycetes -> class:Planctomycetia -> order:Planctomycetales -> no rank:unclassified Planctomycetales -> no rank:unclassified Planctomycetales (miscellaneous) 7 0.02% 255 Finest Classification Taxonomy # % species:uncultured Prosthecobacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Verrucomicrobia -> class:Verrucomicrobiae -> order:Verrucomicrobiales -> family:Verrucomicrobiaceae -> genus:Prosthecobacter -> no rank:environmental samples 7 0.02% species:Bdellovibrio bacteriovorus Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> subphylum:delta/epsilon subdivisions -> class:Deltaproteobacteria -> order:Bdellovibrionales -> family:Bdellovibrionaceae -> genus:Bdellovibrio 7 0.02% species:Paracoccus sp. TP-Snow-C3 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodobacterales -> family:Rhodobacteraceae -> genus:Paracoccus 7 0.02% species:Rhizobium leguminosarum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhizobiales -> family:Rhizobiaceae -> no rank:Rhizobium/Agrobacterium group -> genus:Rhizobium 7 0.02% species:filamentous bacterium Plant1 Iso8 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> no rank:unclassified Bacteroidetes -> no rank:unclassified Bacteroidetes (miscellaneous) 7 0.02% species:uncultured sludge bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental samples 7 0.02% species:Castellaniella sp. 4.5A2 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Alcaligenaceae -> genus:Castellaniella 6 0.01% species:uncultured Acetobacteraceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Alphaproteobacteria -> order:Rhodospirillales -> family:Acetobacteraceae -> no rank:environmental samples 6 0.01% species:Cytophaga sp. BHI60-57B Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Cytophagaceae -> genus:Cytophaga 6 0.01% species:Catonella sp. canine oral taxon 257 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Lachnospiraceae -> genus:Catonella 6 0.01% species:uncultured Rhodoferax sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> family:Comamonadaceae -> genus:Rhodoferax -> no 6 0.01% 256 Finest Classification Taxonomy # % rank:environmental samples species:Microbacterium halophilum Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Actinobacteria -> class:Actinobacteria -> order:Micrococcales -> family:Microbacteriaceae -> genus:Microbacterium 6 0.01% species:uncultured Clostridia bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> no rank:environmental samples 6 0.01% species:Serratia marcescens Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Gammaproteobacteria -> order:Enterobacteriales -> family:Enterobacteriaceae -> genus:Serratia 6 0.01% species:Rhodocyclus tenuis Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Rhodocyclus 6 0.01% species:Azoarcus indigens Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Rhodocyclales -> family:Rhodocyclaceae -> genus:Azoarcus 6 0.01% species:uncultured Clostridiaceae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Clostridia -> order:Clostridiales -> family:Clostridiaceae -> no rank:environmental samples 6 0.01% species:bacterium 64B4 Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Firmicutes -> class:Bacilli -> no rank:unclassified Bacilli -> no rank:unclassified Bacilli (miscellaneous) 6 0.01% species:uncultured Lentisphaerae bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Chlamydiae/Verrucomicrobia group -> phylum:Lentisphaerae -> no rank:environmental samples 6 0.01% species:Lethal yellowing phytoplasma Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Tenericutes -> class:Mollicutes -> order:Acholeplasmatales -> family:Acholeplasmataceae -> genus:Candidatus Phytoplasma -> species group:16SrIV (Coconut lethal yellows group) 6 0.01% species:Sorghum grassy shoot phytoplasma variant I Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Tenericutes -> class:Mollicutes -> order:Acholeplasmatales -> family:Acholeplasmataceae -> genus:Candidatus Phytoplasma -> no rank:unclassified phytoplasmas 6 0.01% species:uncultured Aquabacterium sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> phylum:Proteobacteria -> class:Betaproteobacteria -> order:Burkholderiales -> no rank:unclassified Burkholderiales -> no rank:Burkholderiales Genera incertae sedis -> genus:Aquabacterium -> no rank:environmental samples 6 0.01% species:bacterium enrichment culture clone Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> no rank:environmental 6 0.01% 257 Finest Classification Taxonomy # % R4-76B samples species:uncultured Flexibacter sp. Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi group -> phylum:Bacteroidetes -> class:Cytophagia -> order:Cytophagales -> family:Cytophagaceae -> genus:Flexibacter -> no rank:environmental samples 6 0.01% species:uncultured Bacteroidetes/Chlorobi group bacterium Bacterial: no rank:cellular organisms -> superkingdom:Bacteria -> superphylum:Bacteroidetes/Chlorobi grou