The Use of Hydrogen Peroxide As a Source of Oxygen for Ammonia Removal in Saturated Vertical Flow Constructed Wetlands

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The Use of Hydrogen Peroxide as a Source of Oxygen for Ammonia Removal in Saturated Vertical Flow Constructed Wetlands

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Recommended Citation Dinakar, Madhuri, "The Use of Hydrogen Peroxide as a Source of Oxygen for Ammonia Removal in Saturated Vertical Flow Constructed Wetlands" (2019). Dissertations and Theses. 100. https://digitalcommons.esf.edu/etds/100

This Open Access Thesis is brought to you for free and open access by Digital Commons @ ESF. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of Digital Commons @ ESF. For more information, please contact [email protected], [email protected]. THE USE OF HYDROGEN PEROXIDE AS A SOURCE OF OXYGEN FOR AMMONIA REMOVAL

IN SATURATED VERTICAL FLOW CONSTRUCTED WETLANDS

Madhuri Dinakar

A thesis submitted in partial fulfilment of the requirements for the Master of Science Degree State University of New York College of Environmental Science and Forestry Syracuse, New York August 2019

Department of Environmental Resources Engineering

Approved by: Wendong Tao, Major Professor JoAnne Ellis, Chair, Examining Committee Lindi J. Quackenbush, Department Chair S. Scott Shannon, Dean, The Graduate School

This study could not have been completed without the help and support of several people. First, I would like to thank my advisor Dr. Wendong Tao for his guidance and willingness to help throughout my research. I am appreciative of his advising which pushed me into becoming the researcher I am today. I thank my committee members Dr. Teng Zeng, Prof. Daley and Dr. Nosa Egiebor for their valuable suggestions on the thesis. Next I express my gratitude to Prof. Daley who encouraged me to participate and present my research at the NYWEA annual conference as well as the CNY NYWEA Spring Technical Conference. I must also thank JoAnne Ellis who despite being retired agreed to chair my defense. I am thankful for the graduate assistantships provided by the Environmental Resources Engineering Department and the Graduate School.

I would like to recognize the assistance of Marlene and Kevin from the Analytical and Technical Services at ESF who immensely helped me determine the experimental setup. I extend my sincere thanks to Dr. Stephen Stehman who helped me better understand the data analysis. Many thanks to my lab peers Fred Ageyman, Alper Bayrakdar, Youl Han, and Joel Requena with whom I would have intellectual discussions to troubleshoot. Your support in continuing my experiments while I was away at conferences is also greatly appreciated.

Friends and family are one of the pillars in life. I am extremely thankful to all my friends for their support and encouragement. They would bring a smile to my face on bad days and give me the enthusiasm needed to start with a fresh mind. Thank you to all my colleagues at the Environmental Resources Engineering Department. I would like to acknowledge my family for their financial and emotional support. Special thanks to Sushant Potdar for his positive morale and his help with graphing. Lastly, I am deeply grateful to my parents for their unconditional love and motivation which brought me to ESF to pursue my master’s and encouraged me to complete it.

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Table of Contents

List of Tables ...... vi List of Figures ...... vii Abstract ...... viii Chapter 1: Introduction ...... 1 Objective ...... 2 Chapter 2: Literature review ...... 4 Types of Constructed Wetlands ...... 4 Ammonia Removal in Treatment Wetlands ...... 5 Oxygen Supply in Wetlands...... 8 Hydrogen Peroxide as a Source of Oxygen ...... 10 Hydrogen Peroxide as a Disinfectant...... 10 The Enzyme Catalase ...... 11 Hydrogen Peroxide Use in Constructed Wetlands ...... 12 References ...... 14 Chapter 3: Use of hydrogen peroxide as a source of oxygen for nitrification in vertical flow constructed wetlands ...... 17 Introduction ...... 17 Materials and Methods ...... 18 Experimental Design...... 18 Wetland Setup and Operation ...... 18 Sampling and Data Analysis ...... 20 Plant Growth Measurement ...... 21 Results and Discussion ...... 21 Effect of Hydrogen Peroxide on Dissolved Oxygen Distribution ...... 21 Temperature and pH ...... 23 Effect of Hydrogen Peroxide on Ammonia Removal ...... 25 Effect of Hydrogen Peroxide on Plant Growth and Nitrogen Assimilation ...... 27 Removal Efficiencies with Hydrogen Peroxide Treatment...... 28 Conclusions ...... 29 References ...... 30 Chapter 4: Future discussion and conclusion ...... 33

Appendix ...... 35 Resume ...... 56

List of Tables

Table 3.1. Concentrations of various constituents in synthetic wastewater made to mimic secondary effluent…………………………………………………………………………………… 19 Table 3.2. Operational parameters used during the experiment…………………………………….. 20 Table 3.3. Dissolved oxygen and temperature measurements for various hydrogen peroxide treatments……………………………………………………………………………………………. 22 Table 3.4. Plant growth details for the two wetlands W1 and W2………………………………….. 27

List of Figures

Figure 2.1 Cyperus alternifolius (umbrella sedge) which is a hydrophyte commonly used for SSF wetlands……………………………………………………………………………………………... 7 Figure 2.2. Biochemical reactions involved in nitrification……………………………………….... 9

Figure 2.3. Graph depicting the two bactericidal concentrations of H2O2 ………………………….. 12 Figure 3.1. Experimental setup…………………………………………………………………….... 19 Figure 3.2. Influent flow rates for wetlands W1 and W2…………………………………………… 23 + - Figure 3.3. Results of (a) dissolved oxygen (b) pH variation (c) NH4 -N data (d) NO3 -N values for W1 and W2……………………………………………………………………………………… 24 Figure 3.4. Total Ammonia removal rates and contribution by plant uptake in Wetlands W1 and W2……………………………………………………………………...... 26 Figure 3.5. Regression analysis of height and biomass of C.alternifolius…………………………... 27 Figure 3.6. Change of above-ground plant biomass for the three treatments.………………………. 28 Figure 3.7. Variation in mass removal efficiency and removal efficiency for W1 and W2……….... 28

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Abstract

M. Dinakar. The Use of Hydrogen Peroxide as a Source of Oxygen in Saturated Vertical Flow Constructed Wetlands, 64 pages, 4 tables, 7 figures, 2019. APA style guide used. Nitrification is one of the mechanisms to remove ammonium in constructed wetlands. An important factor affecting this biochemical process is the concentration of dissolved oxygen. This study proposed using hydrogen peroxide as a source of oxygen to ensure enough dissolved oxygen in saturated vertical flow constructed wetlands. Hydrogen peroxide is decomposed into water and oxygen in the presence of catalase in constructed wetlands. Three hydrogen peroxide concentrations, 0.6%, 1% and 2% (w/v) in the dosing solutions, were tested over three periods. The effects of hydrogen peroxide concentration on ammonia removal were assessed in terms of oxygen generation and potential impacts of hydrogen peroxide solution concentration on microorganisms and wetland plants. It was found that as hydrogen peroxide dosing concentration increased, concentration of nitrate in the effluent decreased while an increase in plant growth was observed. It was concluded that the use of hydrogen peroxide affected nitrification by shifting the primary ammonia removal mechanism from nitrification to plant uptake. A dosing solution of 1% was suggested since the greatest plant growth was measured and a uniform dissolved oxygen distribution was measured.

Keywords: Hydrogen peroxide, nitrification, constructed wetland

M. Dinakar Candidate for the degree of Master of Science, August 2019 Wendong Tao, PhD Department of Environmental Resources Engineering State University of New York College of Environmental Science and Forestry Syracuse, New York

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Chapter 1: Introduction

Constructed wetlands have long been used for wastewater treatment due to their sustainable and cost effective treatment approach as compared to conventional wastewater treatment plants (Vymazal, 2010). Constructed wetlands work like natural wetlands by removing or immobilizing excess nutrients. One of the most common nutrients found in wastewater is ammonia. Ammonia is the term used to refer to the + combination of ionized (NH4 ) and unionized (NH3) forms of ammonia, with the dominant species being pH and temperature dependent. Ammonia is synthesized for commercial fertilizers and other industrial processes. Some industrial processes which use ammonia include metal treating and finishing, production of pharmaceuticals and dyes and crude oil processing. Decomposition of organic matter, and atmospheric gas exchange are some examples of natural sources of ammonia (US EPA, 2013). Anthropogenic sources such as municipal and industrial effluent as well as stormwater discharge contain ammonia which enter aquatic environments and increase ammonia concentration within them.

Nitrogen is an essential nutrient required by all organisms to synthesize amino acids. Its oxidation state varies from -3 in ammonia to +5 in nitrate. Changes from one oxidation state to another occur by biochemical reactions in the natural environment. Nitrogen is assimilated by cells in the form of ammonia. Though ammonia is a vital nutrient at high concentrations it becomes toxic to aquatic life. The concentration which results in toxicity is species dependent and has a wide range from 1.8 mg/L to 17 mg/L at pH 7 and 20°C (Randall & Tsui, 2002; US EPA, 2013). The US. EPA reports from 1984 and 1989 (US EPA, 1985,

1989) indicated that 2.79 mg NH3/L was the average acute toxicity threshold for 32 freshwater species measured, while saltwater species could tolerate only 1.86 mg/L. The latest US EPA report (US EPA, 2013) considers ammonia toxicity on other freshwater species such as unionid mussels and gill-breathing snails, based on which the acute and chronic limits were reduced to 1.9 mg/L and 17 mg/L respectively. As seen by the US EPA reports, ammonia is a contaminant which is very strictly regulated, requiring its removal from effluents before it is discharged to surface water.

Eutrophication is another reason requiring strict effluent discharge standards for ammonia. Eutrophication occurs when there’s a surplus of nutrients in the water body (carbon dioxide, phosphorus and nitrogen). All four nutrients are equally important; determining the limiting factor for algal bloom depends on the water body and its characteristics. Eutrophication in lakes and reservoirs is limited mostly by phosphorus, while in marine environments nitrogen is the limiting factor (Yang, Wu, Hao, & He, 2008). Eutrophication causes deterioration in appearance, odor problems arising from the decomposition of algae and hypoxic conditions which can adversely affect aquatic life. Oxygen depletion can occur because of eutrophication as well as nitrification in the receiving water. Oxygen demand exerted by nitrogen for the biological oxidation of

1 ammonia into nitrate will add to the biochemical oxygen demand which could result in anoxic environments (US EPA, 1975)

Disinfection of water/wastewater becomes less effective with the presence of ammonia. The addition of chlorine/ hypochlorite produces chloramines which have a lower effectiveness in disinfection than chlorine/hypochlorite. The presence of ammonia increases the breakpoint concentration and hence the cost of reagents during chemical disinfection (Metcalf & Eddy et al., 2004).

The use of constructed wetlands is one method by which ammonia is treated from wastewater. Constructed wetlands (CW) are treatment systems which utilize natural processes by incorporating wetland vegetation, porous media and the accompanying microbial communities. Since the time constructed wetlands were first used to treat water in the 1950s (Vymazal, 2010), they have evolved into several types with the two primary ones being free water surface (FWS) and subsurface flow (SSF) wetlands. Free water surface wetlands are more comparable to natural wetlands - they have large regions of open water and water depth is relatively shallow. In this type of wetland, water flows slowly over the ground through the aquatic plants wherein the plant roots act as substrates for the microorganisms to grow. Subsurface flow wetlands are wetlands wherein the wastewater flows beneath the surface, through the packing media. Based on the direction of flow, subsurface flow wetlands may further be divided into horizontal flow and vertical flow. With subsurface wetlands comes the disadvantage of low dissolved oxygen (DO) maintained in the wetlands. To increase dissolved oxygen concentration, wetlands need to be aerated by using energy demanding methods. In recent years several other classifications have developed which consider the type of vegetation, saturation extent, and position of loading apart from direction of flow. The performance of contaminant removal varies with the CW design. In natural as well as constructed wetlands, ammonia is removed from water by one of three major mechanisms - plant uptake, adsorption by the packing material/soil, and microbial conversion of nitrogenous compounds into nitrogen gas.

Objective

This study proposes an alternative to conventional aeration strategies. The research objective for this study was to test the use of different concentrations of hydrogen peroxide as a source of oxygen in saturated vertical flow wetlands and thereby analyze its effect on ammonia removal.

References:

Metcalf & Eddy, Tchobanoglous, G., Stensel, H. D., Tsuchihashi, R., Burton, F. L., Franklin L., Abu-Orf, M., … Pfrang, W. (2004). Wastewater engineering : treatment and resource recovery.

Randall, D. & Tsui, T. K. (2002). Ammonia toxicity in fish. Marine Pollution Bulletin, 45(1–12), 17–23. https://doi.org/10.1016/S0025-326X(02)00227-8

US. EPA. (1985). US EPA Ambient Water Quality Criteria for Ammonia-1984. Retrieved from https://www.epa.gov/sites/production/files/2019-02/documents/ambient-wqc-ammonia-1984.pdf

US. EPA. (1989). Ambient Water Quality Criteria for Ammonia (Saltwater)-1989.

US EPA. (1975). Process Design Manual for Nitrogen Control. Retrieved from https://nepis.epa.gov/Exe/ZyNET.exe/9100PUPQ.TXT?ZyActionD=ZyDocument&Client=EPA&In dex=Prior+to+1976&Docs=&Query=&Time=&EndTime=&SearchMethod=1&TocRestrict=n&Toc =&TocEntry=&QField=&QFieldYear=&QFieldMonth=&QFieldDay=&IntQFieldOp=0&ExtQField Op=0&XmlQuery=&

US EPA, O. (2013). Aquatic Life Criteria - Ammonia. Retrieved from https://www.epa.gov/wqc/aquatic- life-criteria-ammonia

Vymazal, J. (2010). Constructed wetlands for wastewater treatment. Water, 2(3), 530–549. https://doi.org/10.3390/w2030530

Yang, X., Wu, X., Hao, H., & He, Z. (2008). Mechanisms and assessment of water eutrophication. Journal of Zhejiang University SCIENCE B, 9(3), 197–209. https://doi.org/10.1631/jzus.B0710626

Chapter 2: Literature review

Types of Constructed Wetlands

Constructed wetlands are mainly of two types- free water surface (FWS) wetlands and subsurface flow (SSF) wetlands. An FWS wetland is typically a shallow basin with 20-40 cm of water above 20-30 cm of rooting substrate. The vegetation can be emergent, floating or submerged plants. Wastewater is treated by processes of sedimentation, filtration, adsorption and biochemical reactions. The open water in FWS wetlands increases the risks of water borne vectors such as mosquitos. To avoid possible human exposure to pathogens, FWS wetlands are rarely used for secondary treatment, instead these wetlands are used for tertiary treatment or advanced treatment processes. Free water surface flow wetlands treat several types of contaminants; however, their overall efficiency reduces in cold climates in open water. The layer of ice formed acts as a barrier to oxygen transfer and hinders oxygen dependent biochemical processes such as nitrification. Removal of organics had been found to be more efficient under cold climates compared to warmer summers. FWS wetlands are frequently used to treat stormwater because of their robustness against changing water levels and pulse flows. Subsurface flow wetlands consist of a media bed over an impermeable liner planted with macrophytes. In horizontal subsurface flow (HSSF) wetlands, wastewater flows across the wetlands through the pores of the media and between the plant roots. Biochemical treatment occurs in the biofilm formed around the media with an oxygen gradient present within the biofilm. Subsurface flow systems are more expensive than FWS wetlands in terms of capital cost with the operational costs being similar. Unlike FWS wetlands, SSF wetlands are commonly used for secondary treatment in small communities or single-family homes. Advantages compared to FWS wetlands include the elimination of the risk of public contact and water-borne vectors as well as a better cold climate tolerance. Horizontal surface flow wetlands have the disadvantage of maintaining a low dissolved oxygen (DO) concentration due to saturated conditions maintained in the wetland which results in long hydraulic retention time (HRT) in large wetlands to meet the required effluent standards. To overcome this, vertical flow wetlands were designed (Kadlec & Wallace, 2008).

Vertical flow (VF) wetlands are another kind of subsurface flow wetland. In Europe these wetlands were developed as a solution to the low DO problem in HSSF wetlands. Oxygen availability is increased in VF wetlands due to intermittent feeding and drainage wherein aerobic conditions are restored in the system. In North America VF wetlands are normally designed as vegetated gravel filters. During intermittent feeding, large batches of influent are fed at once causing a temporary flooding of the surface with 3-5cm of water which then percolates through the packing material and is treated. The medium bed is drained completely

4 before it is fed again, allowing air to refill the wetland. Due to the higher DO levels that they maintain VF wetlands require less land and shorter HRTs which translates into lower construction costs. The operation of VF wetlands by feeding and draining leads to a major problem of clogging. Therefore, the packing material needs to be judiciously selected and the system needs to be designed to maintain an optimum hydraulic loading rate (Stefanakis, Akratos, & Tsihrintzis, 2014; Vymazal, 2011). VF wetlands have also been used to create anaerobic conditions in the bottom of the bed to foster Sulphur reducing bacteria by maintaining a thick water column (Kadlec & Wallace, 2008)

The most common setup for a vertical flow is a 0.45-1.2 m deep (Stefanakis et al., 2014) wetland which is covered in the bottom with an impermeable liner to prevent from seepages. The wetland is normally filled with gravel/sand and has a slight slope of 1-2% at the bottom to allow for water to be drained. Since intermittently loaded VF wetlands can achieve high DO levels, they have been used to treat ammonia rich wastewater such as dairy wastewater, landfill leachate and food processing wastewater (Kadlec & Wallace, 2008). One disadvantage of operating the wetland as intermittent feeding is the small contact time between the contaminant and the microorganisms; other modes of operation such as recirculation and tidal flow increase contact times. In tidal flow water is pumped upwards through the bed until the bed is saturated. The wetland is kept saturated for a while before it is drained which allows for air to re-establish aerobic conditions. This mode of operation also ensures that aerobic conditions are maintained constantly. Recirculation is wherein part of the effluent is recirculated to increase the contact time while also diluting the influent thereby achieving an improved organic matter removal performance. The last type of operation is maintaining the wetlands at saturated condition, which are operated either as up flow or downflow. Saturated conditions allow for longer retention times, thereby allowing longer contact times and hence better removal of contaminants. Due to poor DO levels in saturated wetlands, other mechanisms of external aeration techniques need to be employed. This study was focused on saturated downflow vertical flow wetlands.

Ammonia Removal in Treatment Wetlands

+ - The inorganic forms of nitrogen seen in treatment wetlands include ammonia (NH4 ), nitrite (NO2 ), nitrate - (NO3 ), nitrous oxide (N2O) and lastly dissolved elemental nitrogen. Treatment wetlands may also contain organic forms of nitrogen which include amino acids, purines, pyrimidines, urea and uric acid. The proportions of organic and inorganic forms of nitrogen depend on the source of water being treated as well as the type of wetland being used. It has been reported that domestic wastewater has 60% inorganic nitrogen and the rest 40% as organic nitrogen and less than 1% as nitrite-nitrate (US EPA, 1975). These fractions change with wastewater sources, for example food processing effluents have a very high organic nitrogen

5 concentration. Effluents from lagoons and FWS wetlands also tend to have a greater organic nitrogen fraction. In treatment wetlands, both dissolved and particulate nitrogen forms can be present, though particulate nitrogen forms are lower in settled waters (Kadlec & Wallace, 2008). Among the dissolved and particulate forms, dissolved nitrogen forms have the highest impact on water bodies since they can be consumed by microorganism leading to eutrophication and a net decrease in dissolved oxygen (DO) in the water body.

+ Among the inorganic nitrogenous species excess in ammonia nitrogen (NH4 -N) leads to eutrophication and other associated water quality issues. In constructed wetlands, ammonia is treated by three leading mechanisms; plant uptake, adsorption by the packing material, and microbial reactions. Ionized ammonia in the wastewater can get adsorbed onto the packing material in SSF and soil in FWS wetlands. The amount of ammonia adsorbed depends on the water chemistry (e.g. if nitrification reduces ammonia concentrations, ammonia will be desorbed to maintain equilibrium) and the type of packing media being used. Since the ammonium ion is positively charged, it is subject to cation exchange. Gravels which are commonly used in SSF wetlands have a lower cation exchange capacity (CEC) than natural zeolites, meaning adsorption on gravel would be lesser. Marble chips have been found to have an even lower adsorption capacity for ammonia than the common mixture of gravel (Tao, Wen, & Huchzermeier, 2011). Organic sediments which are present in FWS wetlands have a CEC between natural zeolites and gravel. Exposing the wetland to air by intermediate feeding could result in the oxidation of adsorbed ammonia to nitrate which is not bound to the substrate. It has been reported the adsorption of ammonia can be modelled with the Freundlich equation (Kadlec & Wallace, 2008; Vymazal, 2007).

Another mechanism involved in ammonia removal is plant uptake. Plants in constructed wetlands play an integral role in contaminant removal of organics, inorganics and metals. For ammonia, the average removal rate by the vegetative cycle (assimilation, growth followed by death/decay) is 10 g N/m2-yr (Kadlec & Wallace, 2008). This amount may be insignificant for systems with high loading rates however for systems with lower loading, plants can contribute a large portion of the ammonia removal. Plants can consume ammonia and nitrate with the preference depending on the respective concentrations as well as the plant species. This said, the ammonia preference is more common among macrophytes since it is more reduced than nitrate and is energetically easier to assimilate into amino acids, unless the water is ammonia-poor in which case nitrate becomes the predominant nitrogen source (Lee, Fletcher, & Sun, 2009; Vymazal, 2007). A study performed by Zhu and Sikora in 1994 (Sikora, Tong, Behrends, Steinberg, & Coonrod, 1995) showed that 70-85% of entire nitrate loss in SSF systems containing no carbon source and no ammonia in the effluent was due to plant uptake. It has been noted that at lower pH the nitrate uptake is favored and at neutral pH ammonia is preferred nitrogen source (Havlin, Tisdale, Nelson, & Beaton, 2014).

The plant which was used in this study is Cyperus alternifolius (Fig. 2.1) which is also commonly referred to as umbrella sedge. It is a hydrophytic plant which can tolerate broad pH and temperature ranges.

A study published in 2018 (Tao, 2018) provides details about plant growth rate (above-ground), nitrogen content in above and below ground biomass individually and nitrogen assimilation rates by Cyperus alternifolius for VF constructed wetlands. Umbrella sedge was found to have slower biomass growth rates in VF wetlands (at 0.42-4.4 g (dw)/m2/day) as compared to FWS wetlands (6.6-33.1 g(dw)/m2/day). The nitrogen content for above ground and below ground tissues was found to be 34.3 mg N/g and 14.3 mg N/g respectively. Lastly the nitrogen assimilation rates were found to be 0.27–0.94 g/m2/day.

Fig. 2.1 Cyperus alternifolius (umbrella sedge) which is a hydrophyte commonly used for SSF wetlands The last and most common ammonia removal mechanism observed is removal through microbial action. Microorganisms convert ammonium into nitrogenous compounds such as nitrates, nitrites and nitrogen gas through processes such as the familiar nitrification-denitrification process (wherein nitrification is a two- step process), simultaneous nitrification-denitrification and ANAMMOX (anaerobic ammonia oxidation). These pathways have been known to occur in several combinations (based on environmental conditions). The discovery of ANAMMOX bacteria and the versatility of ammonia oxidizing bacteria led to the development of new nitrogen removal techniques such as SHARON (Single reactor system for High-rate Ammonium Removal Over Nitrite), CANON (Completely Autotrophic Nitrogen removal Over Nitrite), and OLAND (Oxygen-Limited Autotrophic Nitrification-Denitrification)

Nitrifying bacteria are typically aerobic and chemoautotrophic in nature; they oxidize ammonium to nitrate - in a two-step process. Initially ammonium is oxidized to nitrite (NO2 ) which is then further oxidized to nitrate. The first reaction is brought about by ammonia oxidizing bacteria (AOB) from genera Nitrosospira, Nitrosococcus and Nitrosomonas (Eq. 1) and is commonly referred to as nitritation. Nitrite oxidizing bacteria (NOB) comprising of bacteria from genera Nitrospira and Nitrobacter participate in oxidizing nitrite to nitrate (Eq. 2) which is known as nitratation (Lee et al., 2009).

+ − + 2NH4 + 3O2 → 2NO2 + 2H2O + 4H Eq. 1

− − 2NO2 + O2 → 2NO3 Eq. 2 As with most microbial reactions, nitrification too is an enzyme catalyzed process. Nitritation is a two stepped enzymatic process; the first step in an endergonic reaction (ΔG°= +17 kJmol-1), which is catalyzed by the cytoplasmic membrane bound enzyme ammonia monooxygenase for which the substrate is NH3 + rather than the commonly perceived NH4 . Oxygen is required as molecular oxygen and electrons from the ubiquinone-cytochrome b part of the electron transport chain are consumed to result in hydroxylamine

(NH2OH) as the product (Eq. 3)

+ − NH3 + O2 + 2H + 2e → NH2OH + H2O Eq. 3

- The next reaction involves the oxidation of NH2OH to NO2 which is exergonic in nature. The periplasmic enzyme hydroxylamine oxidoreductase oxidizes NH2OH with oxygen from water to result in the formation - of NO2 with four electrons being released (Eq. 4). The overall nitritation reaction is exergonic with ΔG°= -271 kJ/mol NH3-N.

− + − NH2OH + H2O → NO2 + 5H + 4e Eq. 4

Of the four electrons released by hydroxylamine, two are consumed for the first reaction by ammonia monooxygenase and the remaining two electrons enter the electron transport chain, thereby producing the required energy for biosynthesis (Prosser, 1990). The production of nitrate from nitrite is less exergonic than the synthesis of nitrite (ΔG°=-78 kJ/mol NH3-N) (Halling-Sørensen & Jørgensen, 1993), meaning the growth rate of bacteria responsible for nitratation is lower (doubling time = 12-59 hr.) than the growth rate of nitritation bacteria (doubling time= 8-36 hr.) (Hiet Wong, Barton, & Barford, 2003). The enzyme nitrite oxidoreductase is responsible for the oxidation of nitrite into nitrate (Fig. 2.2)

Oxygen Supply in Wetlands

Based on equations 1 and 2 it’s evident that oxygen is required for nitrification; according to the + stoichiometry, 4.57 g O2/gNH4 -N is required. Considering biomass synthesis, only 4.3 g of oxygen is consumed with an alkalinity requirement of 7.14 mg/L as CaCO3. Having DO (dissolved oxygen) levels less than 1-2 mg/L has proven to substantially reduce nitrification rates (Hammer & Knight, 1994) with an optimum DO level being 3 mg/L (US EPA, 1975).

Fig. 2.2. Biochemical reactions involved in nitrification

If bacteria in the constructed wetlands do not acquire sufficient oxygen, the efficiency of ammonia removal will drastically reduce. In constructed wetlands, particularly FWS wetlands oxygen is made available to the microorganisms through three ways- surface aeration, plant oxygen transport and photosynthesis (EPA, 2000). Since the area of open water available in SSF wetlands is minute, the major mechanism for supplying oxygen is photosynthesis and plant oxygen transport. However, plant oxygen transport can only aerate the rhizosphere meaning the deeper regions of the SSF wetland (0.5-0.9m (Kadlec & Wallace, 2008)) remain anaerobic posing a difficulty for oxygen requiring biochemical reactions such as nitrification.

To compensate for lower DO levels in the wetland, external aeration mechanisms need to be employed such as blowers, pumps, and compressors (Kadlec & Wallace, 2008). These external aeration methods are very energy intensive. One study published by Austin and Nivala investigated the power consumption of aerated wetlands and found it to be 485 kWh/d with the estimate for modified Ludzack–Ettinger activated sludge treatment process being 884 kWh/d (Austin & Nivala, 2009). Because of this high energy demands, other techniques have been used to supply the necessary oxygen such as intermittent feeding (Fan, Wang, et al., 2013; Fan, Zhang, et al., 2013), tidal flow and recirculation of the effluent (Foladori, Ruaben, & Ortigara, 2013). Non-aerated systems have an ammonia removal efficiency ≥ 59% while aerated systems have been reported to have a removal efficiency ≥ 90% (Saeed & Sun, 2012). Apart from increasing DO and improving nutrient removal efficiencies, aerated wetlands have also been proven to have lower greenhouse gas emissions particularly methane and nitrous oxide (Maltais-Landry, Maranger, & Brisson, 2009) and that they perform better than non-aerated systems in cold climates (Ouellet-Plamondon, Chazarenc, Comeau, & Brisson, 2006; Saeed & Sun, 2012). Despite these facts and figures, the addition of an aeration does not guarantee increased performance since the efficiency of aeration depends on the oxygen transfer rates (OTR). Oxygen transfer rate refers to the rate at which oxygen can be transferred from the aerators to

9 the wetlands. This depends on bed depth, interstitial oxygen concentration, diffuser geometry along with other factors (Kadlec & Wallace, 2008). Other technologies have also been used recently to enhance DO levels in the water. For example, a recent procedure employed is to treat the waste water in an algal pond before it enters a constructed wetland (Ding et al., 2016) . In this study, it was observed that the algal pond helped in increasing the DO content and hence aided in nitrification. Another group of researchers tried using a wind powered air blower as an aerator in constructed wetlands (Boog et al., 2016). The accompanying cost for forced aeration as well as the fact that DO levels in saturated VF wetlands are poor necessitate new aeration methods to be developed.

Hydrogen Peroxide as a Source of Oxygen

Hydrogen peroxide is a common metabolic product formed in most organisms caused by oxidation- reduction reactions during the electron transport chain for energy production. It is synthesized as an intermediate in catabolic processes wherein oxygen is used to oxidize organic carbon into carbon dioxide and water simultaneously releasing energy. The amount of hydrogen peroxide produced is small and is immediately decomposed by enzymes due to its cytotoxic nature in high concentrations (Schumb, Satterfield, & Wentworth, 1955). In bacteria, the primary enzyme involved in the decomposition of hydrogen peroxide is catalase (Callow, 1923). Catalase catalyzes the decomposition of H2O2 to result in water and oxygen according to Eq. 5. It should be noted that catalase is found in nearly all bacteria including nitrifying bacteria particularly ammonia oxidizing bacteria (AOB) (Wood & Sørensen, 2001).

catalase 2H2O2 → O2 + 2H2O Eq. 5

From the equation, it is seen that two moles of hydrogen peroxide (68g) produce one mole of oxygen (32g) upon dissociation. By mass this would mean 1g would result in 0.47g of oxygen.

Hydrogen Peroxide as a Disinfectant Commonly hydrogen peroxide has been used in water and wastewater treatment as a disinfectant and advanced oxidizing agent. However, its capacity to disinfect is quite low compared to other chemicals such as chlorine, chlorine dioxide or ozone (Aslani et al., 2014), hence a high concentration of H2O2 is required to achieve the disinfection standards for drinking water, if not it has to be supported with other chemicals. 2+ 2+ Recent research has found that the addition of metallic cations (Ag or Fe ) along with H2O2 significantly improves disinfection (Aslani et al., 2014). Advanced oxidation processes (AOP) have been developed which include treatments like H2O2 with O3 and H2O2 with UV (Deng & Zhao, 2015). The commercial formulation of hydrogen peroxide is either 35% or 50% by weight. In drinking water treatment, the dosage used to disinfect ranges from 50 mg/L to 250 mg/L based on the dosing interval and the concentration of

10 hydrogen peroxide used (the commercial formulation) (Cotruvo, Craun, & Hearne, 1999; Safety Hydrogen peroxide as an oxidizing agent Use of hydrogen peroxide, n.d.). The mechanism of disinfection is by hydroxyl radicals that are produced when hydrogen peroxide reacts with the superoxide radical (Eq. 6); first proposed by Haber and Weiss (Haber & Weiss, 1934). The hydroxyl radicals are then responsible for causing oxidative stress which in turn destroys the cell.

− − O2 • + H2O2 → O2 + OH + OH • Eq. 6

The bactericidal nature of hydrogen peroxide occurs by two modes; mode-1 and mode-2 as shown in Fig. 2.3. At concentrations <3mM the first level of bactericidal activity is seen as a slight dip. If hydrogen peroxide concentrations are further increased no change in bactericidal activity was observed until 20mM. Beyond 20mM irreversible damage occurs to the cell and the cell suffers cell death. The two modes of cell death have been found to be due to different mechanisms; mode-1 is due to DNA nicking caused by H2O2 which is dependent on Ferryl radical intermediates from DNA complexation. This said, at higher hydrogen peroxide concentrations the DNA associated iron becomes a limiting factor, therefore further increase in hydrogen peroxide concentrations will not cause DNA damage at the rate expected. The innate DNA repair mechanisms allow cells to overcome the stress caused by mode-1 disinfection. The mechanism for mode- 2 is complete oxidation of other biomolecules in the cell membrane such as proteins and lipids, which is irreversible leading to the cell membrane becoming unstable and ultimately cell death (Linley, Denyer, McDonnell, Simons, & Maillard, 2012).

The Enzyme Catalase

Since hydrogen peroxide degrades to oxygen in the presence of catalase containing microorganisms, it has been used as a source of supplemental oxygen for many applications, one of them being for wastewater treatment which will be discussed further in Chapter 3. The ability of hydrogen peroxide to degrade into water and oxygen depends on catalase activity. Majority of catalases found in bacteria and plants are monofunctional catalases which are made up of hemeproteins. Some plant catalases may have a specific nonpolypeptide unit bound at the catalytic center. The monofunctional catalases found in plants are expressed predominantly in photosynthetic tissues (Sharma, 2014). The decomposition of hydrogen peroxide into water and oxygen has two distinct stages. The first stage is wherein the heme iron of the enzyme gets oxidized by H2O2 into an intermediate complex. During this stage the oxygen-oxygen bond in peroxides is cleaved heterolytically with one oxygen molecule leaving as water and the other still in the intermediate complex. Reduction of the heme-intermediate complex by one more hydrogen peroxide molecule is the second stage of the reaction, which ends with the release of oxygen and water. Therefore,

11 the catalytic reaction pathway has H2O2 acting as an oxidant initially and later as a reductant. These reactions are displayed as Eqs 7-8 (Zamocky, Furtmüller, & Obinger, 2008).

Fig. 2.3. Graph depicting the two bactericidal concentrations of H2O2 (Linley et al., 2012)

•+ Por Fe(III) + H2O2 → Por Fe(IV) = O + H2O Eq. 7

•+ Por Fe(IV) = O + H2O2 → Por Fe(III) + H2O + O2 Eq. 8

Studies have shown that the catalytic reaction of breaking down hydrogen peroxide into water and oxygen is a first order reaction with a reaction rate coefficient in the order of 107 per second (Aebi, 1974; Sepasi Tehrani & Moosavi-Movahedi, 2018; Zamocky et al., 2008) at low hydrogen peroxide concentrations which at greater concentrations switches to a zero order reaction (Jones & Suggett, 1968; Northrop, 1925). Until a hydrogen peroxide concentration of 0.1M the reaction follows Michaelis-Menten enzyme kinetics with the reaction rate increasing with increase in substrate concentration. Beyond 0.1M there is rapid inactivation of catalase which is dependent on reaction/exposure time (Aebi, 1974; Jones & Suggett, 1968).

The inactivation of catalase when hydrogen peroxide concentration is greater than 0.1M H2O2 switches the reaction to a zero-order reaction wherein substrate concentration does not affect the rate of the reaction.

Hydrogen Peroxide Use in Constructed Wetlands

The addition of hydrogen peroxide into CWs would affect other components of the wetland as well such as the bacteria and plants. Hydrogen peroxide has been used to aerate soils which helps in root growth.

Therefore, the addition of H2O2 in the wetlands would enhance root growth. Increase in germination was also observed and was attributed to an increase in ascorbic acid degrading enzymes in the seed (Ismail, Khandaker, Mat, & Boyce, 2015). For the nitrifying bacterial community the addition of hydrogen peroxide at high concentrations would be detrimental since hydrogen peroxide inhibits the enzyme hydroxylamine - oxidoreductase involved in the oxidation of NH2OH to NO2 (Hooper & Terry, 1977).

References

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Chapter 3: Use of hydrogen peroxide as a source of oxygen for nitrification in vertical flow constructed wetlands

Introduction Oxygen is required for nitrification in all types of constructed wetlands including vertical flow constructed wetlands (VFCWs). The saturation concentration of dissolved oxygen at 20°C is 9.1 mg/L. When the dissolved oxygen level in wastewater is below 1-2 mg/L, it substantially reduces nitrification rates (Hammer & Knight, 1994). One of the oxygenation methods is to chemically supply oxygen. This study focused on using hydrogen peroxide as an oxygen source in saturated wetlands.

Chin and Hicks ( 1970) evaluated the technique of using H2O2 to increase the DO content in BOD tests for wastewater. The tests showed that using H2O2 increased the dissolved oxygen content while not disturbing the micro-fauna. Young and Baumann (1973) concluded that small quantities of hydrogen peroxide could be added as an instant source of oxygen in activated sludge. Cole et al. (1974) compared a hydrogen peroxide treated system and a diffused-air aeration system in bench-scale experiments and found that there was no statistical difference pertaining to the removal of organics and that there were vast differences in nitrification. Ammonia removal efficiency decreased from 90% to 17% with H2O2 concentrations increasing from 40mg/L to 440mg/L in the dosing solution despite no difference seen below 40 mg/L. Houtmeyers et al (1977) found that addition of hydrogen peroxide into the reactor to maintain a DO of

4mg/L ± 0.5 mg/L using 3% H2O2, decreased ammonium oxidizers by nearly 85% while nitrite oxidizers remained constant throughout the study. A greater catalytic activity was observed within the microorganisms for the hydrogen peroxide treated system as compared to the compressed air aerated system.

Despite these adverse impacts of hydrogen peroxide solutions on nitrification, a recent publication claimed that addition of hydrogen peroxide enhances ammonium removal from domestic wastewater (Jóźwiakowski et al., 2017). This study showed that as oxygen levels increased due to addition of hydrogen peroxide, ammonium removal efficiencies increased from 39% to 81.2%, the most efficient being when the concentration of dissolved oxygen level was 30-40% of oxygen saturation levels. Hydrogen peroxide has its application in constructed wetlands as a strong oxidizing agent to reduce clogging in subsurface flow wetlands (Nivala & Rousseau, 2009). In constructed wetlands, another factor involved is vegetation. Hydrogen peroxide has been used to aerate roots to enhance plant growth (Walter et. al 2004; Ismail et al. 2015). An increase in growth would allow for greater areas for biofilm formation in constructed wetlands.

The different effects on nitrification in the earlier studies may be due to different H2O2 concentrations used to dose the systems, which ranged from 3% to 35% dosing solutions in the studies showing deleterious

effects on nitrification, and 0.1% in the research performed by Jóźwiakowski et al. (2017). Also, the previous work has been done to simulate conventional wastewater treatment facilities, none of which have focused on constructed wetlands that have plants. This study aimed to use different hydrogen peroxide concentrations to maintain an aerobic environment and study its effect on ammonia removal in saturated vertical flow constructed wetlands.

Materials and Methods Experimental Design Two vertical flow wetlands (mesocosms) were used to test the effect of using hydrogen peroxide as a source of oxygen, using three different concentrations of hydrogen peroxide solutions: 0.6%, 1% and 2% while maintaining a constant mass flow rate of H2O2. The flow rates of the hydrogen peroxide dosing solutions were set to ensure a DO level of 3 mg/L or greater in the wetlands. To account for variations in DO with depth, DO was measured at the point of feeding and at the bottom of the wetland. Experiments were performed in two stages, with an initial preliminary stage to verify the operational parameters which lasted from September 2018 to December 2018 and then upon modification of conditions, the experiment was conducted from December 2018 to May 2019. To ensure minimum temperature influences, the wetlands were placed in a greenhouse where temperature was assumed to be relatively constant across the treatments.

The concentration of hydrogen peroxide used in the earlier studies was as high as 3-35% along with the DO maintained at as high as 4-7 mg/L. The recent study conducted by Jóźwiakowski et al. (2017) used a 0.1% solution to maintain DO at different saturation levels. The pharmaceutically accepted concentration of hydrogen peroxide to act as a disinfectant is 3% (PubChem, ID:784). Given that and the studies previously conducted, these three concentrations were chosen.

Wetland Setup and Operation Two vertical flow wetlands made of polyvinyl chloride pipes were filled with previously used marble chips (between sieves 2 mm and 4.75 mm) to achieve a porosity of 0.5 (Fig. 3.1). Porosity was measured by filling a 1-L cylinder with the sieved marble chips and adding water until the 1L mark. Water was then poured into a cylinder to measure the volume. Each of the pipes had a total height of 75 cm and an inner diameter of 15.2 cm with the marble chips being filled to a height of 60 cm. Marble chips are a commercially available product. Being composed of calcium carbonate, marble chips have been used as a packing material of constructed wetlands especially for their property of supplementing alkalinity to buffer pH (Tao, Wen, & Norton, 2011; Wen, Tao, Wang, & Pei, 2013). The VFCWs were planted with Cyperus alternifolius (common name: umbrella sedge). Hydrogen peroxide was pumped in the middle through the sampling port at the edge of the wetland using a Fischer Scientific Manostat peristaltic pump.

Fig.3.1. Experimental setup. 1 and 2: Sampling ports.

Microbial biomass attached to polystyrene beads were collected from a biological aerated filter for ammonia removal at Metropolitan Syracuse Wastewater Treatment Plant. The beads were immersed in tap water and sonicated at 30kHz with a power output of 300W for 5 × 1 min to dislodge biofilms, which were used to inoculate the wetlands. Solids analysis of the biomass suspension showed that the inoculum had an average total solids content of 1.196 g/L and a VS content of 0.273 g/L. Each wetland was seeded with 1.3 L of the inoculum and feeding of the wastewater began the next day. Samples for DO, pH, ammonia and nitrite-nitrate were taken 3 HRTs after feeding to allow for acclimatization.

Synthetic wastewater was used as the influent during this study. The wastewater was made to resemble secondary effluent. This stage in the treatment process was chosen since CWs are normally used as tertiary treatment systems (Vymazal, 2011). Table 3.1 illustrates the typical ranges for the predominant elements in wastewater along with the concentrations used in this study.

Table 3.1. Concentrations of various constituents in synthetic wastewater made to mimic secondary effluent.

+ Element NH4 -N Total P Cl Na K Ca Mg Fe Alkalinity

(as CaCO3) Rangea (mg/L) 0.1-45 5-40 50-500 50-400 10-30 25-100 10-50 0-0.7 0.39-200 Concentration 40 10 75 55 15 50 20 0.35 70.8 (mg/L) a: obtained from (Metcalf & Eddy et al., 2004) and National Research Council,, 1996)

Batches of synthetic wastewater were made by dissolving salts NH4Cl, K2HPO4, CaCl2, MgSO4, Na2CO3 and FeSO4• 7H2O into de-chlorinated tap water. These salts were chosen to ensure all the elements are within the typical ranges and to ensure maximum solubility while also taking into consideration macronutrients and predominant micronutrients required for plant and microbial growth.

The two VFCWs were designed as plug flow systems with the reaction coefficient corresponding to nitrification in vertical flow wetlands taken as 0.3 d-1 (Abdelhakeem, Aboulroos, & Kamel, 2016). Equation 1 was used to determine initial hydraulic retention time (HRT).

ka q = C Eq. 1 ln i Ce

+ Initial conditions for the study were modelled with an influent NH4 -N concentration of 20 mg/L and HRT of 4 d. However, since the nitrate levels were negligible in tests using 0.6% and 1% H2O2 dosing solutions, the influent concentration as well as HRT were altered. The HRT was changed to 2 d and influent concentration to 40 mg/L. The design and operational parameters are specified in Table 2.

Table 3.2. Operational parameters used during the experiment Parameter Units Value Height of marble chips packed cm 60 Bulk volume of packed bed m3 0.01 3 -3 Effective volume (Veff) m 5.44x10 HRT d 2 Flow rate (influent) ml/min 1.8 Hydraulic loading rate m/d 0.147 Flow rate of 0.6% H2O2 every 30 minutes for ml/min 0.6 1 minute Flow rate of 1.0% H2O2 every 30 minutes for ml/min 0.3 1 minute Flow rate of 2.0% H2O2 every 30 minutes for ml/min 0.15 1 minute

Sampling and Data Analysis Water samples were collected every two days (HRT) for approximately 20 days for each hydrogen peroxide solution concentration at the middle and bottom of each wetland. For the samples in the bottom, 200 ml of water was drained then 50 ml was collected as the sample. Similarly, for the samples in the middle, 100ml was drained and 50 ml was collected. Draining water before collecting samples was to flush the lines and thereby obtain representative samples. The difference in volume of water flushed was due to the difference in the lengths of the lines between bottom and middle. After samples were collected, dissolved oxygen (DO), temperature and pH were measured using Fischer portable meters. Effluent volumes were measured

20 every two days to calculate flow rate. Samples collected were analyzed for ammonia and nitrate+nitrite using the QuickChem 8500 automatic flow injection analyzer (LaChat Instrument, Loveland, CO). Ammonia analysis was performed using the phenolate method while the hydrazine reduction method was employed to determine nitrite+nitrate concentration. A chemical oxygen demand analysis was conducted on 10th December 2018 to verify low C/N ratios. A mass balance of nitrogen was performed to understand the relative contribution of different nitrogen removal mechanisms for the different hydrogen peroxide concentrations used. The total mass removal rate of ammonia minus the total accumulation rate of nitrate was considered to be plant nitrogen assimilation rates, assuming only nitrification and plant assimilation of ammonia and nitrate determined the fate of nitrogen in the wetlands. ANOVA was used to test at α=0.05 whether the concentrations of hydrogen peroxide solution had a significant effect on ammonia removal. At the end of the study, biofilm on the marble chips was measured by collecting grab samples at top, middle and bottom and performing a volatile solids analysis.

Plant Growth Measurement Initial plant height was recorded before the wetlands were treated with the first concentration of hydrogen peroxide. After every dose treatment, plant height was measured by using a tape to measure the height of the plant from 1cm above the media to the tip of the leaves. The difference between initial and final plant height was used to obtain the change in height. At the end of the test for all the concentrations of hydrogen peroxide, plants were uprooted and separated into above- and below-ground tissues, and individual plant height and biomass was recorded to establish a relationship between height and above-ground biomass as well as above- to below-ground biomass ratio. The regression equations and ratio were used retroactively to estimate the biomass initially and after the first and second periods with the measured plant heights.

Results and Discussion Wetlands 1 and 2 were operated as plug flow systems and this was verified by calculating the Reynolds number. The Reynolds number was 0.003 for both wetlands for all three treatments which is much below the limit of laminar flow Reynolds number of 1 (Kadlec and Wallace, 2008). Based on these results other calculations were made considering the systems to be plug flow. Effect of Hydrogen Peroxide on Dissolved Oxygen Distribution ANOVA was conducted with concentration of H2O2 as a variable. It was found that both wetlands showed a significant variation of DO saturation levels (W1: p=0.001 and W2: p=0.004) with hydrogen peroxide dosing concentration. As shown in Table 3.3, both wetlands showed the same pattern wherein DO in the bottom was greatest for 1% followed by the 0.6% treatment and lastly the 2% treatment. When a Fisher test was conducted for DO values pertaining to the bottom for both the wetlands, it was seen that there existed a statistical difference between all DO values for W1 while only a statistical difference between 1% and 2% existed for W2. The DO values in the middle were different with the lowest being at 0.6% treatment.

In both the wetlands the difference between 1% and 2% treatments was insignificant for the dissolved oxygen concentration in the middle. Considering both the wetlands, for the 0.6% treatment the saturation level in the middle was half that of the bottom, for the 1% treatment the distribution was more uniform with no significant difference between the middle and bottom and lastly for the 2% treatment the middle was significantly greater than the bottom.

The rate of H2O2 decomposition with respect to the flow rate could explain the differences in DO levels with different H2O2 concentrations. Several studies have reported that at H2O2 concentrations below 0.1 M

(0.34% w/w of 100% H2O2) the decomposition reaction in the presence of catalase is a first order reaction, meaning the rate is proportional to substrate concentration (Aebi, 1974; Northrop, 1925; Sepasi Tehrani &

Moosavi-Movahedi, 2018). At the low H2O2 concentration of 0.6% the reaction rate would be slower than the flow rate, meaning the decomposition would predominantly occur in the bottom of the wetland. As the concentration of hydrogen peroxide increased it balanced out against flow rate and at 2% was greater or equal to the flow rate resulting in the reaction predominantly occurring in the middle of the wetland.

Table 3.3 Dissolved oxygen and temperature measurements for various hydrogen peroxide treatments

H2O2 Treatment Wetland Location Sample size DO (% saturation) Temperature °C 0.6 W1 Middle 12 40.61 ± 4.76 18.4 ± 2.5 W1 Bottom 12 89 ± 3.27 18.4± 2.3 W2 Middle 8 49.81 ± 9.15 18.5 ± 2.9 W2 Bottom 8 90.98 ± 16.58 18.6 ± 3.0 1.0 W1 Middle 11 93.28 ± 15.37 19.8 ± 1.9 W1 Bottom 11 101.90±8.34 19.8±1.8 W2 Middle 9 99.76 ± 6.41 18.5 ± 1.5 W2 Bottom 11 104.97 ± 19.81 19.7 ±1.8 2.0 W1 Middle 10 94.59 ± 6.87 20.0 ± 1.6 W1 Bottom 10 77.84±7.73 20.9±2.5 W2 Middle 8 95.13 ± 4.23 19.4 ± 2.2 W2 Bottom 9 78.61± 6.19 20.4± 1.9

Another reason which could explain the DO pattern among the three concentrations is the inhibition of catalase by nitrite and nitrate (Krych-Madej & Gebicka, 2017). It was found that inhibition of catalase by nitrite was greater in the presence of chloride ions and at pH 7.4 compared to pH 5. Since this system was - designed as a plug-flow reactor the effluent concentrations were bound to have greater NO3 -N than

22 concentrations in the middle. Hence since the inhibition of catalase follows Michaelis-Menten kinetics, with greater nitrite/nitrate concentrations, a greater number of enzyme units get inhibited, resulting in lower DO production. It was also found that oxygen release by hydrogen peroxide decomposition was greatest at

1.5% H2O2 (George, 1949). Lastly the presence of plant roots near the middle of the wetlands would also contribute to the greater DO concentrations with increased H2O2 concentration. Plant oxygen transfer has 2 also been noted to vary greatly from 0.005-12g O2/m -d (Nivala et al., 2013). Based on the average influent and effluent flow rates measured (Fig. 3.2.), it was estimated that oxygen transfer rate was approximately 2 2 4.1 g O2/m /d from H2O2 decomposition and 1.2 g O2/m /d from influent. Comparing the estimated oxygen transfer rates with ammonia mass removal rates (Fig. 3.4.) suggested that direct assimilation of ammonium by plants played a significant role in ammonia removal, especially in the periods with 1.0% and 2.0% H2O2 + - dosing solutions. Plants grown in NH4 -N media compared to NO3 -N media contain greater levels of catalase in their roots (Medici, Azevedo, Smith, & Lea, 2004). Since the primary source of nitrogen in the influent was NH4Cl, catalase production would be greater in the roots resulting in more oxygen near the middle. Lastly it has also been noted that the decomposition of hydrogen peroxide may not be stoichiometric and result in greater oxygen production since the reaction depends on the amount of catalase present (Northrop, 1925; Sergius Morgulis, 1921). The above stated reasons along with the fact that the roots are closer to the middle could account for the DO pattern observed in both the wetlands.

Fig. 3.2 Influent flow rate for the 2 wetlands over the three treatments. The design flow rate was 1.8ml/min Temperature and pH The variation seen in temperature and pH was noted to be the same in both the wetlands. Temperature did not statistically depend on the concentration of hydrogen peroxide dosed. For both the wetlands, mean temperatures increased as the test progressed from 0.6% to 2% (Table 3.3). The increase in temperature could be accounted for by seasonal variation from December to May. Regarding pH, it was found that concentration did not statistically influence pH for W1 and W2 p=0.061 and p=0.355. Means of pH and

23 other along with the associated variation is depicted in Fig 3.3. A decrease in pH was observed between the influent to effluent pH which could demonstrate nitrification.

It is known that changes in temperature will not alter the activity of catalase since the enzyme has a very low activation energy (600-1700 cal/mol) (Aebi, 1974). This said, the influent temperature was relatively constant at 20.13°C. It has been reported that catalase activity varies minimally in the pH range of 4 to 8.5 (Britton Chance, 1952; Zamocky, Furtmüller, & Obinger, 2008), hence it can be concluded that the small differences in pH did not cause differences in catalase activity. Regarding nitrification, it is widely accepted that nitrification is accelerated at higher temperatures, however this refers only to suspended growth systems. Since this system has fixed films, the effect of temperature is minor since the more influencing factor becomes diffusion processes across the biofilm (Chen, Ling, & Blancheton, 2006). The optimal pH range for nitrification depends on the class of bacteria considered (Chen et al., 2006), with the accepted range being 7-9 wherein nitrification rates decrease significantly when pH<5.5 (US EPA, 1975). Based on the above cited literature it can be concluded that variations in temperature and pH did not have a significant effect on nitrification nor decomposition of hydrogen peroxide.

(a) (b)

Fig. 3.3. Results of (a) dissolved oxygen measured at B-bottom and M-middle, (b) effluent pH, (c) + - effluent NH4 -N concentration, and (d) effluent NO3 -N concentration of Wetlands W1 and W2 dosed with 0.6%, 1.0% and 2.0% hydrogen peroxide solutions.

Effect of Hydrogen Peroxide on Ammonia Removal Ammonia nitrogen values for all three concentrations of hydrogen peroxide decreased from 40 mg/L in influent to less than 6 mg/L in W1 effluent and less than 3 mg/L in W2 effluent. For the 0.6% treatment, in W1 33% of the data points were below method detection limit (0.05 mg/L) while 12.5% of data was below 0.05 mg/L in W2. In the 1% treatment, W1 and W2 had 27% and 72% of their data below 0.05. Lastly for 2% treatment, 90% of W1 data and all the W2 data were ≤ 0.05 mg/L. Values below IDL were replaced with IDL concentrations for statistical analysis. This shows that there is a difference in effluent ammonia concentrations among the three treatments; the ANOVA results also indicate the same p=0.017 for W1 and p=0.011 for W2 (Appendix A). The difference in means between W1 and W2 for the same treatments can be accounted for by the difference in plant density and thereby plant assimilation of nitrogen as well as the surface area available for biofilm formation- this is seen in the VS results wherein W2 had a greater biomass + compared to W1.These variations in NH4 -N concentrations agree with the statistical significance that hydrogen peroxide concentration had on effluent nitrate concentrations p<0.001 for both W1 and W2.

Nitrate concentrations decreased with increasing H2O2 concentrations as seen in Fig 3.3. with the difference between all concentrations being statistically significant in W1 while W2 showed a statistical difference between 0.6% and 1% and 0.6% and 2%. The inhibition of hydroxylamine oxidoreductase by hydrogen - peroxide could be one reason for the reduction in NO3 -N values other reasons are increased nitrification rates and greater plant assimilation rates.

+ Although the effluent NH4 -N was maintained at ≤ 6mg/L and ≤ 3mg/L in W1 and W2 respectively, nitrate concentration was not increased equally, meaning another process of ammonia treatment was dominating which is plant uptake (Fig 3.4). Figure 3.4 depicts the proportion of ammonia removal due to plant assimilation as mass removal rates. Plant assimilation rates were calculated based on the difference between + - + total mass of NH4 -N in the system and residual NO3 -N as NH4 -N. Since it was unclear which form of nitrogen plants were consuming, a definite estimation of nitrification rate could not be made. The green part in the graph shows the proportion of plant uptake with the total being represented by the bar height. As seen, the proportion of plant assimilation increased with greater hydrogen peroxide concentrations. This shows that there was a shift in primary ammonia removal mechanism. The reported rate for nitrogen assimilation by Cyperus alternifolius is 0.11–0.25 g/m2-day (Tao, 2018) which would have increased in this scenario due to increased growth rate caused by the addition of hydrogen peroxide as discussed in the - next section. Another reason for reduced NO3 -N values is similar; high DO concentration would result in + increased nitrification rates which would reduce the availability of NH4 -N for plants forcing them to utilize - NO3 -N as the source of nitrogen. Nitrification rates in wetlands have been reported to be in the range 0.01- 2.15 g/m2/d with a mean value of 0.048 g/m2/d (Reddy & D’Angelo, 1997; Tanner, Kadlec, Gibbs, Sukias, & Nguyen, 2002; Vymazal, 2007). The values calculated are shown in Fig 3.4 are much higher than mean

25 values proving that nitrification rates could have increased. It is generally noted in literature that nitrification rates increase until 4mg DO/L beyond which it is independent of DO (Chen et al., 2006; Stenstrom & Poduska, 1980; US EPA, 1975). Zhang et al (1996) found that the DO in the biofilm was much lower than bulk water due to a diffusion resistance (Chen et al., 2006). Since the system being studied has fixed film growth, though the DO levels much higher than 4mg/L in the system, within the biofilm it can be much lower. As DO concentrations increased in the middle with increasing H2O2 dosing solution concentrations, the DO within the biofilm would increase, thereby causing an increase in reaction rate.

This idea of greater reaction rates is more probable since the minimum H2O2 concentration needed for inhibition is greater than the concentrations tested. This is further proven by the fact that as DO values increased statistically in the middle from 43% saturation in the 0.6% treatment to 94% saturation in the other two treatments in W1 and from 49.8% to 99.7% in W2, nitrate concentration in the effluent reduced from 36.33 mg/L to 15.97 mg/L in W1 and 39.61 to 15.88 mg/L in W2.

Fig. 3.4. Total Ammonia removal rates and contribution by plant uptake in Wetlands W1 and W2 dosed with 0.6%, 1.0% and 2.0% hydrogen peroxide solutions, respectively.

- The difference between NO3 -N concentrations in the 1% and 2% treatments for W2 was statistically insignificant which coincide results obtained for DO. Hence it can be inferred that majority of nitrification was occurring in the middle of the wetland where H2O2 was dosed. Biofilm measurements taken at the end of the study proved this with VS content decreasing in this order: Top>Middle>Bottom. The VS values measured for samples collected in the top, middle and bottom were 3.53E-05, 2.04E-05 and 2.06E-05 g VS/ g marble chips for W1 while W2 presented values of 3.05E-05, 5.07E-05 and 3.93E-05 g VS/ g marble chips respectively. .

Effect of Hydrogen Peroxide on Plant Growth and Nitrogen Assimilation The relationship of height to dry biomass was found to be an exponential relationship as seen in Fig 3.5. The average height, number of plants and above biomass growth rate is presented in Table 3.4. The below ground to above ground ratio was determined to be 0.535 for W1 and 0.506 for W2. This is greater than the values previously reported for C. alternifolius (Tao, 2018), which could be attributed to increased growth rate due to the application of hydrogen peroxide. Increased plant growth with hydrogen peroxide + concentrations resulted in greater NH4 -N assimilation depicted in in Fig. 3.6 and Fig. 3.4. Above ground as well as below ground biomass increased with increase in H2O2 concentration over a span of approximately 50 days thereby clarifying that the contribution of plant uptake increased with increasing

H2O2.

Table 3.4. Plant growth details for the two wetlands W1 and W2

W1 W2 0.6%: Average density (# plants/m2) 4797 6433 Average height (cm) 63 57.97 Above ground growth rate g (dw)/m2-day - 43.89 1%: Average density (# plants/m2) 5643 10214 Average height (cm) 65.07 59.17 Above ground growth rate g (dw)/m2-day 43.25 101.58 2%: Average density (# plants/m2) 8352 12359 Average height (cm) 69.69 61.41 Above ground growth rate g (dw)/m2-day 87.47 92.55

Since growth rate was increasing with hydrogen peroxide concentrations, nitrogen assimilation rate would + increase, thereby affecting effluent nitrate concentration. With greater plant assimilation rates, NH4 -N - available for nitrification is reduced as well as lower NO3 -N levels in the effluent, resulting in decreasing - NO3 -N with H2O2. This was also seen with the nitrate concentrations for W1 and W2.

Fig. 3.5. Regression analysis of height and biomass of C. alternifolius.

Fig. 3.6. Change of above-ground plant biomass for the three treatments.

Removal Efficiencies with Hydrogen Peroxide Treatment Both MRe (mass removal efficiency) and Re (concentration reduction efficiency) increased with increasing + H2O2 (Fig. 3.7). This shows that addition of H2O2 accelerated NH4 -N removal as previously noted

(Jóźwiakowski et al., 2017). Areal mass removal rates (MRRa) for 0.6, 1 and 2% hydrogen peroxide dosing 2 solution treatments for W1 were 4.25, 5.23, 5.78 g/m /d. For W2 MRRa for the three treatments were 5.35, 4.89, 5.74 g/m2/d. The volumetric mass removal rates were calculated to be 13.85, 17.06, 18.85 g/m3/d for W1 and 17.45, 15.94, and 18.69 corresponding to W2 for 0.6%, 1% and 2% treatments respectively.

Fig. 3.7. Variation in mass removal efficiency and removal efficiency for W1 and W2

Conclusions • Ammonia removal was affected by concentration of hydrogen peroxide dosing solution. At greater ammonia concentrations, nitrification rate and plant growth increased, resulting in a better performance • Overall all the wetlands were maintained aerobic, however variations were seen in the DO at middle

and bottom among the three concentrations of H2O2. The possible reasons for this pattern were attributed to reaction rate kinetics, inhibition of catalase by nitrite and plant oxygen release. Plant oxygen transfer increased with increasing hydrogen peroxide concentration.

+ • Effluent NH4 -N was reduced to lesser than 6mg/L in W1 and 3mg/L in W2 from approximately + 38mg/l. For the 0.6% H2O2 treatment 58% of influent NH4 -N was accounted for by nitrification in W1 and 33% in W2. As the concentration of hydrogen peroxide increased, rate of nitrification - increased forcing plants to consume NO3 -N as their sole source of nitrogen. Further kinetic studies would be needed to prove this. Based on DO measurements it was inferred that majority of nitrification was occurring in the middle

• Plant growth increased with increasing H2O2 with above-ground:below-ground ratios being greater than previously estimated. Plant biomass increase was greatest for the 1% treatment with the increase being 51% in W1 and 69% in W2. As concentration of hydrogen peroxide dosing solution increased, a shift in primary removal mechanism of nitrogen was observed.

• Based on the results obtained, 1% H2O2 could be used as a suitable source of oxygen in vegetated saturated constructed wetlands. This concentration allowed for nitrification to proceed at its maximum and ensured a uniform DO distribution.

References Abdelhakeem, S. G., Aboulroos, S. A., & Kamel, M. M. (2016). Performance of a vertical subsurface flow constructed wetland under different operational conditions. Journal of Advanced Research, 7(5), 803–814. https://doi.org/10.1016/J.JARE.2015.12.002 Aebi, H. (1974). Catalase. In Methods of Enzymatic Analysis (pp. 673–684). Elsevier. https://doi.org/10.1016/B978-0-12-091302-2.50032-3 Britton Chance. (1952).Effect of pH upon the reaction kinetics of the enzyme-substrate compounds of catalse. The Journal of Biological Chemistry. Retrieved from http://www.jbc.org/content/194/2/471.citation Chen, S., Ling, J., & Blancheton, J.-P. (2006). Nitrification kinetics of biofilm as affected by water quality factors. Aquacultural Engineering, 34(3), 179–197. https://doi.org/10.1016/J.AQUAENG.2005.09.004 Chin, C., & Hicks, M. G. (1970). Hydrogen Peroxide Studies of Oxygen Demand. Journal (Water Pollution Control Federation), 42(7), 1327–1342. Retrieved from http://www.jstor.org/stable/25036713 Cole, C. A., Ochs, L. D., & Funnell, F. C. (1974). Hydrogen Peroxide as a Supplemental Oxygen Source. Journal (Water Pollution Control Federation), 46(11), 2579–2592. Retrieved from http://www.jstor.org/stable/25038306 George, P. (1949). The effect of the peroxide concentration and other factors on the decomposition of hydrogen peroxide by catalase. The Biochemical Journal, 44(2), 197–205. https://doi.org/10.1042/bj0440197 Hammer, D. A., & Knight, R. L. (1994). Designing Constructed Wetlands for Nitrogen Removal. Water Science and Technology, 29(4), 15–27. Retrieved from http://wst.iwaponline.com/content/29/4/15 Houtmeyers, J., Poffé, R., & Verachtert, H. (1977). Hydrogen peroxide as a supplemental oxygen source for activated sludge: Microbiological investigations. European Journal of Applied Microbiology and Biotechnology, 4(4), 295–305. https://doi.org/10.1007/BF00931267 Ismail, S. Z., Khandaker, M. M., Mat, N., & Boyce, A. N. (2015). Effects of Hydrogen Peroxide on Growth, Development and Quality of Fruits: A Review. Journal of Agronomy, 14(4), 331–336. https://doi.org/10.3923/ja.2015.331.336 Jóźwiakowski, K., Marzec, M., Fiedurek, J., Kamińska, A., Gajewska, M., Wojciechowska, E., … Kowlaczyk-Juśko, A. (2017). Application of H2O2 to optimize ammonium removal from domestic wastewater. Separation and Purification Technology, 173, 357–363. https://doi.org/10.1016/j.seppur.2016.08.047 Krych-Madej, J., & Gebicka, L. (2017). Interactions of nitrite with catalase: Enzyme activity and reaction kinetics studies. Journal of Inorganic Biochemistry, 171, 10–17. https://doi.org/10.1016/j.jinorgbio.2017.02.023 Medici, L. O., Azevedo, R. A., Smith, R. J., & Lea, P. J. (2004). The influence of nitrogen supply on antioxidant enzymes in plant roots. Functional Plant Biology, 31(1), 1. https://doi.org/10.1071/FP03130 Metcalf & Eddy, Tchobanoglous, G., Stensel, H. D., Tsuchihashi, R., Burton, F. L. (Franklin L., Abu-Orf, M., … Pfrang, W. (2004). Wastewater engineering : treatment and resource recovery.

National Research Council (U.S.). Committee on the Use of Treated Municipal Wastewater Effluents and Sludge in the Production of Crops for Human Consumption. (1996). Use of reclaimed water and sludge in food crop production. National Academy Press. Nivala, J., & Rousseau, D. P. L. (2009). Reversing clogging in subsurface-flow constructed wetlands by hydrogen peroxide treatment: two case studies. Water Science and Technology, 59(10), 2037–2046. https://doi.org/10.2166/wst.2009.115 Nivala, J., Wallace, S., Headley, T., Kassa, K., Brix, H., van Afferden, M., & Müller, R. (2013). Oxygen transfer and consumption in subsurface flow treatment wetlands. Ecological Engineering. https://doi.org/10.1016/j.ecoleng.2012.08.028 Northrop, J. H. (1925).The kinetics of the decomposition of peroxide by catalase. The Journal of General Physiology, 7(3), 373–387. https://doi.org/10.1085/jgp.7.3.373 PubChem Database, National Center for Biotechnology Information PubChem Database. Hydrogen peroxide, CID=784, https://pubchem.ncbi.nlm.nih.gov/compound/Hydrogen-peroxide (accessed on July 28, 2019) Reddy, K. R., & D’Angelo, E. M. (1997). Biogeochemical indicators to evaluate pollutant removal efficiency in constructed wetlands. Water Science and Technology, 35(5), 1–10. https://doi.org/10.1016/S0273-1223(97)00046-2 Sepasi Tehrani, H., & Moosavi-Movahedi, A. A. (2018). Catalase and its mysteries. Progress in Biophysics and Molecular Biology, 140, 5–12. https://doi.org/10.1016/j.pbiomolbio.2018.03.001 Sergius Morgulis. (1921).A study of the catalase reaction. Journal of Biological Chemistry, 47, 341–375. Retrieved from http://www.jbc.org/ Stenstrom, M. K., & Poduska, R. A. (1980). The effect of dissolved oxygen concentration on nitrification. Water Research, 14(6), 643–649. https://doi.org/10.1016/0043-1354(80)90122-0 Tanner, C. C., Kadlec, R. H., Gibbs, M. M., Sukias, J. P. S., & Nguyen, M. L. (2002). Nitrogen processing gradients in subsurface-flow treatment wetlands—influence of wastewater characteristics. Ecological Engineering, 18(4), 499–520. https://doi.org/10.1016/S0925- 8574(02)00011-3 Tao, W. (2018). Microbial removal and plant uptake of nitrogen in constructed wetlands: mesocosm tests on influencing factors. Environmental Science and Pollution Research, 25(36), 36425–36437. https://doi.org/10.1007/s11356-018-3543-4 Tao, W., Wen, J., & Norton, C. (2011). Laboratory study on factors influencing nitrogen removal in marble chip biofilters incorporating nitritation and anammox. https://doi.org/10.2166/wst.2011.679 US EPA. (1975). Process Design Manual for Nitrogen Control. Retrieved from https://nepis.epa.gov/Exe/ZyNET.exe/9100PUPQ.TXT?ZyActionD=ZyDocument&Client=EPA&In dex=Prior+to+1976&Docs=&Query=&Time=&EndTime=&SearchMethod=1&TocRestrict=n&Toc =&TocEntry=&QField=&QFieldYear=&QFieldMonth=&QFieldDay=&IntQFieldOp=0&ExtQField Op=0&XmlQuery=& Vymazal, J. (2007). Removal of nutrients in various types of constructed wetlands. Science of The Total Environment, 380(1–3), 48–65. https://doi.org/10.1016/J.SCITOTENV.2006.09.014 Vymazal, J. (2011). Constructed Wetlands for Wastewater Treatment: Five Decades of Experience. Environmental Science & Technology, 45(1), 61–69. https://doi.org/10.1021/es101403q

Walter, S., Heuberger, H., & Schitzler, W. H. (2004).Sensibility of different vegetables to oxygen deficiency and aeration with H2O2 in the rhizosphere. Acta Horticulturae, (659), 499–508. https://doi.org/10.17660/ActaHortic.2004.659.66 Wen, J., Tao, W., Wang, Z., & Pei, Y. (2013). Enhancing simultaneous nitritation and anammox in recirculating biofilters: Effects of unsaturated zone depth and alkalinity dissolution of packing materials. Journal of Hazardous Materials, 244–245, 671–680. https://doi.org/10.1016/J.JHAZMAT.2012.10.058 Young, J. C., & Baumann, E. R. (1973). Hydrogen peroxide aids in measuring sludge oxygen uptake rates. Zamocky, M., Furtmüller, P. G., & Obinger, C. (2008). Evolution of Catalases from Bacteria to Humans. Antioxidants & Redox Signaling, 10(9), 1527–1548. https://doi.org/10.1089/ars.2008.2046 Zhang, T. C., & Bishop, P. L. (1996). Evaluation of substrate and pH effects in a nitrifying biofilm. Water Environment Research, 68(7), 1107–1115. https://doi.org/10.2175/106143096X128504

Chapter 4: Future discussion and conclusion

In this study, the effect of hydrogen peroxide concentration on ammonia removal in saturated vertical flow wetlands was investigated. The results obtained showed that ammonia removal was influenced by hydrogen peroxide dosed. Plant assimilation of nitrogen increased with concentration of hydrogen peroxide. It was unclear which form of nitrogen the plants were assimilating. Ammonia removal efficiencies increased with greater hydrogen peroxide concentration reaching nearly 100% removal at 2% dosing solution concentration. This removal efficiency is much higher than the removal efficiencies observed for constructed wetlands without aeration i.e. 50-60% (Kadlec & Wallace, 2008; Vymazal, 2010). However, when supplied with oxygen either by air or chemicals, removal efficiency increases to 85% or greater (Lee, Fletcher, & Sun, 2009). The current study agrees with past literature.

The results showed that when a 1% (w/v) H2O2 dosing solution was used, dissolved oxygen concentration was uniformly distributed in the wetland. The increase in plant assimilation was also greatest at 1% H2O2. Therefore, among the three concentrations tested 1% hydrogen peroxide can be used to supply oxygen in saturated wetlands. Saturated vertical flow wetlands were used in this study; however, the results are also applicable to horizontal sub surface flow wetlands.

Based on a cost estimate previously conducted for using hydrogen peroxide as a source of oxygen in horizontal flow sand filters, it was estimated that the capital cost of using hydrogen peroxide was greater than using aerators. However, when operational costs were considered, the use of hydrogen peroxide was cheaper (Jóźwiakowski et al., 2017). This agrees with another study performed on a sand bed filter wherein

0.6% H2O2 was used to maintain a dissolved oxygen level of 7mg/L. The authors found that the installation of controlling devises would be the most expensive (Müller & Sekoulov, 2013).

Hydrogen peroxide is an explosive chemical at concentrations of 35% and greater since it decomposes exothermically into water and oxygen. Therefore, transportation of high concentrations is not a viable solution. Instead, hydrogen peroxide can be synthesized onsite using the water purified by electrolysis (Drogui, Elmaleh, Rumeau, Bernard, & Rambaud, 2001). Research has also shown that waste disinfection hydrogen peroxide is more effective at oxidation compared to the pure chemical (Bhatti et al., 2011).

With this information, the capital cost of hydrogen peroxide would be lower than calculated. Given this and the results obtained in the current study, dilute concentrations hydrogen peroxide can be used as a source of oxygen in constructed wetlands.

References:

Bhatti, Z. A., Mahmood, Q., Raja, I. A., Malik, A. H., Rashid, N., & Wu, D. (2011). Integrated chemical treatment of municipal wastewater using waste hydrogen peroxide and ultraviolet light. Physics and Chemistry of the Earth, Parts A/B/C, 36(9–11), 459–464. https://doi.org/10.1016/J.PCE.2010.03.024

Drogui, P., Elmaleh, S., Rumeau, M., Bernard, C., & Rambaud, A. (2001). Hydrogen peroxide production by water electrolysis: Application to disinfection. Journal of Applied Electrochemistry, 31(8), 877– 882. https://doi.org/10.1023/A:1017588221369

Jóźwiakowski, K., Marzec, M., Fiedurek, J., Kamińska, A., Gajewska, M., Wojciechowska, E., … Kowlaczyk-Juśko, A. (2017). Application of H2O2 to optimize ammonium removal from domestic wastewater. Separation and Purification Technology, 173, 357–363. https://doi.org/10.1016/j.seppur.2016.08.047

Kadlec, R. H., & Wallace, S. (2008). Treatment Wetlands, Second Edition. CRC Press. Retrieved from https://books.google.com/books?id=hPDqfNRMH6wC

Lee, C., Fletcher, T. D., & Sun, G. (2009). Nitrogen removal in constructed wetland systems. Engineering in Life Sciences, 9(1), 11–22. https://doi.org/10.1002/elsc.200800049

Müller, W. R., & Sekoulov, I. (2013). Intensification of the ammonia oxidation in sandbed filter. In S. H. Jenkins (Ed.), Proceedings of the Conference on Nitrogen As a Water Pollutant (pp. 483–493). Pergamon. Retrieved from http://www.sciencedirect.com/science/article/pii/B978148321344650037X

Vymazal, J. (2010). Constructed Wetlands for Wastewater Treatment. Water, 2(3), 530–549. https://doi.org/10.3390/w2030530

Appendix Wetland 1: One-way ANOVA: %DO versus Concentration

Method

Null hypothesis All means are equal Alternative Not all means are equal hypothesis Significance level α = 0.05

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

Concentration 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Concentration 2 3048 1523.8 9.84 0.001 Error 30 4646 154.9 Total 32 7693

Model Summary

S R-sq R-sq(adj) R-sq(pred)

12.4443 39.61% 35.59% 27.64%

Means

Concentration N Mean StDev 95% CI

0.6 12 89.00 17.61 (81.66, 96.34)

1.0 11 101.90 8.34 (94.24, 109.56) 2.0 10 77.84 7.73 (69.80, 85.88)

Pooled StDev = 12.4443

Fisher Pairwise Comparisons

Grouping Information Using the Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

1.0 11 101.90 A 0.6 12 89.00 B 2.0 10 77.84 C

Means that do not share a letter are significantly different.

Wetland 2 One-way ANOVA: %DO versus Concentration

Method

Null hypothesis All means are equal Alternative Not all means are equal hypothesis Significance level α = 0.05

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

Concentration 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Concentration 2 3465 1732.7 7.04 0.004 Error 25 6156 246.2 Total 27 9621

Model Summary

S R-sq R-sq(adj) R-sq(pred)

15.6917 36.02% 30.90% 20.48%

Means

Concentration N Mean StDev 95% CI

0.6 8 90.98 16.58 (79.55, 102.40) 1.0 11 104.97 19.81 (95.23, 114.72) 2.0 9 78.61 6.19 (67.84, 89.38)

Pooled StDev = 15.6917

Fisher Pairwise Comparisons

Grouping Information Using the Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

1.0 11 104.97 A 0.6 8 90.98 A B 2.0 9 78.61 B

Means that do not share a letter are significantly different.

Wetland 1 One-way ANOVA: Temp versus Concentration

Method

Null hypothesis All means are equal Alternative Not all means are equal hypothesis Significance level α = 0.05

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

Concentration 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Concentration 2 35.89 17.944 3.66 0.038 Error 30 147.27 4.909 Total 32 183.15

Model Summary

S R-sq R-sq(adj) R-sq(pred)

2.21559 19.59% 14.23% 2.57%

Means

Concentration N Mean StDev 95% CI

0.6 12 18.397 2.306 (17.091, 19.703) 1.0 11 19.844 1.755 (18.480, 21.208) 2.0 10 20.940 2.538 (19.509, 22.371)

Pooled StDev = 2.21559

Fisher Pairwise Comparisons

Grouping Information Using the Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

2.0 10 20.940 A 1.0 11 19.844 A B 0.6 12 18.397 B

Means that do not share a letter are significantly different.

Wetland 2 One-way ANOVA: Temp versus Concentration

Method

Null hypothesis All means are equal Alternative Not all means are equal hypothesis Significance level α = 0.05

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

Concentration 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Concentration 2 14.77 7.384 1.50 0.242 Error 25 122.93 4.917

Total 27 137.70

Model Summary

S R-sq R-sq(adj) R-sq(pred)

2.21749 10.73% 3.58% 0.00%

Means

Concentration N Mean StDev 95% CI

0.6 8 18.58 3.00 (16.96, 20.19) 1.0 11 19.677 1.779 (18.300, 21.054) 2.0 9 20.439 1.880 (18.917, 21.961)

Pooled StDev = 2.21749

Fisher Pairwise Comparisons

Grouping Information Using the Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

2.0 9 20.439 A 1.0 11 19.677 A 0.6 8 18.58 A

Means that do not share a letter are significantly different.

Wetland 1 One-way ANOVA: NH4 versus Concentration

Method

Null hypothesis All means are equal Alternative Not all means are equal hypothesis Significance level α = 0.05

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

Concentration 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Concentration 2 125.0 62.49 4.71 0.017 Error 30 397.7 13.26 Total 32 522.7

Model Summary

S R-sq R-sq(adj) R-sq(pred)

3.64097 23.91% 18.84% 8.65%

Means

Concentration N Mean StDev 95% CI

0.6 12 5.20 4.46 (3.05, 7.35) 1.0 11 4.20 3.91 (1.96, 6.44) 2.0 10 0.585 1.692 (-1.766, 2.936)

Pooled StDev = 3.64097

Fisher Pairwise Comparisons

Grouping Information Using the Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

0.6 12 5.20 A 1.0 11 4.20 A 2.0 10 0.585 B

Means that do not share a letter are significantly different.

Wetland 2 One-way ANOVA: NH4-N versus Concentration

Method

Null hypothesis All means are equal Alternative Not all means are equal hypothesis Significance level α = 0.05

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

Concentration 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Concentration 2 19.46 9.729 5.47 0.011 Error 25 44.50 1.780 Total 27 63.96

Model Summary

S R-sq R-sq(adj) R-sq(pred)

1.33417 30.42% 24.86% 9.53%

Means

Concentration N Mean StDev 95% CI

0.6 8 2.069 2.443 (1.097, 3.040) 1.0 11 0.426 0.522 (-0.403, 1.254) 2.0 9 0.05000 0.00000 (-0.86592, 0.96592)

Pooled StDev = 1.33417

Fisher Pairwise Comparisons

Grouping Information Using the Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

0.6 8 2.069 A 1.0 11 0.426 B 2.0 9 0.05000 B

Means that do not share a letter are significantly different.

Wetland 1 One-way ANOVA: NO3-N versus Concentration

Method

Null hypothesis All means are equal Alternative Not all means are equal hypothesis Significance level α = 0.05 Rows unused 1

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

Concentration 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Concentration 2 2363.0 1181.51 49.46 0.000 Error 29 692.7 23.89 Total 31 3055.8

Model Summary

S R-sq R-sq(adj) R-sq(pred)

4.88749 77.33% 75.77% 72.35%

Means

Concentration N Mean StDev 95% CI

0.6 12 36.32 4.61 (33.44, 39.21) 1.0 10 23.19 5.18 (20.03, 26.35) 2.0 10 15.97 4.91 (12.81, 19.13)

Pooled StDev = 4.88749

Fisher Pairwise Comparisons

Grouping Information Using the Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

0.6 12 36.32 A 1.0 10 23.19 B 2.0 10 15.97 C

Means that do not share a letter are significantly different.

Wetland 2 One-way ANOVA: NO3-N versus Concentration

Method

Null hypothesis All means are equal Alternative Not all means are equal hypothesis Significance level α = 0.05 Rows unused 1

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

Concentration 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Concentration 2 2978 1489.06 18.82 0.000 Error 24 1899 79.12 Total 26 4877

Model Summary

S R-sq R-sq(adj) R-sq(pred)

8.89516 61.06% 57.82% 51.25%

Means

Concentration N Mean StDev 95% CI

0.6 8 39.61 4.97 (33.12, 46.10) 1.0 10 17.34 10.80 (11.54, 23.15) 2.0 9 15.88 9.20 (9.76, 22.00)

Pooled StDev = 8.89516

Fisher Pairwise Comparisons

Grouping Information Using the Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

0.6 8 39.61 A 1.0 10 17.34 B 2.0 9 15.88 B

Means that do not share a letter are significantly different.

Wetland 1 One-way ANOVA: pH versus Concentration

Method

Null hypothesis All means are equal Alternative Not all means are equal hypothesis Significance level α = 0.05 Rows unused 1

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

Concentration 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Concentration 2 0.9915 0.4957 3.09 0.061 Error 29 4.6501 0.1603 Total 31 5.6416

Model Summary

S R-sq R-sq(adj) R-sq(pred)

0.400435 17.57% 11.89% 0.37%

Means

Concentration N Mean StDev 95% CI

0.6 12 6.622 0.496 (6.386, 6.859) 1.0 10 6.460 0.459 (6.202, 6.719) 2.0 10 6.1973 0.0713 (5.9383, 6.4563)

Pooled StDev = 0.400435

Fisher Pairwise Comparisons

Grouping Information Using the Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

0.6 12 6.622 A 1.0 10 6.460 A B

2.0 10 6.1973 B

Means that do not share a letter are significantly different.

Wetland 2 One-way ANOVA: pH versus Concentration

Method

Null hypothesis All means are equal Alternative Not all means are equal hypothesis Significance level α = 0.05 Rows unused 1

Equal variances were assumed for the analysis.

Factor Information

Factor Levels Values

Concentration 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Concentration 2 0.6382 0.3191 1.08 0.355 Error 24 7.0834 0.2951 Total 26 7.7216

Model Summary

S R-sq R-sq(adj) R-sq(pred)

0.543268 8.27% 0.62% 0.00%

Means

Concentration N Mean StDev 95% CI

0.6 8 6.885 0.707 (6.489, 7.281) 1.0 10 6.631 0.628 (6.276, 6.986) 2.0 9 6.5019 0.0712 (6.1281, 6.8756)

Pooled StDev = 0.543268

Fisher Pairwise Comparisons

Grouping Information Using the Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

0.6 8 6.885 A 1.0 10 6.631 A 2.0 9 6.5019 A

Means that do not share a letter are significantly different.

Wetland 1 General Linear Model: %DO versus Location, Concentration

Method

Factor coding (-1, 0, +1)

Factor Information

Factor Type Levels Values

Location Fixed 2 B, M Concentration Fixed 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Location 1 2955 2954.9 23.07 0.000 Concentration 2 12797 6398.6 49.97 0.000 Location*Concentration 2 11953 5976.4 46.67 0.000 Error 60 7683 128.1 Total 65 36343

Model Summary

S R-sq R-sq(adj) R-sq(pred)

11.3162 78.86% 77.10% 74.55%

Regression Equation

%DO = 82.87 + 6.71 Location_B - 6.71 Location_M - 18.07 Concentration_0.6 + 14.72 Concentration_1.0 + 3.35 Concentration_2.0 + 17.49 Location*Concentration_B 0.6 - 2.40 Location*Concentration_B 1.0 - 15.08 Location*Concentration_B 2.0 - 17.49 Location*Concentration_M 0.6 + 2.40 Location*Concentration_M 1.0 + 15.08 Location*Concentration_M 2.0

Fits and Diagnostics for Unusual Observations

Obs %DO Fit Resid Std Resid

4 66.44 89.00 -22.56 -2.08 R 5 65.31 89.00 -23.69 -2.19 R 6 118.59 89.00 29.59 2.73 R 8 112.36 89.00 23.36 2.16 R 47 56.06 93.28 -37.22 -3.45 R

R Large residual

Means

Fitted Term Mean SE Mean

Location B 89.58 1.98 M 76.16 1.98 Concentration 0.6 64.80 2.31 1.0 97.59 2.41 2.0 86.22 2.53 Location*Concentration B 0.6 89.00 3.27 B 1.0 101.90 3.41 B 2.0 77.84 3.58 M 0.6 40.61 3.27 M 1.0 93.28 3.41 M 2.0 94.59 3.58 Comparisons for %DO Fisher Pairwise Comparisons: Location

Grouping Information Using Fisher LSD Method and 95% Confidence

Location N Mean Grouping

B 33 89.5794 A M 33 76.1601 B

Means that do not share a letter are significantly different.

Fisher Pairwise Comparisons: Concentration

Grouping Information Using Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

1.0 22 97.5909 A 2.0 20 86.2150 B 0.6 24 64.8033 C

Means that do not share a letter are significantly different.

Fisher Pairwise Comparisons: Location*Concentration

Grouping Information Using Fisher LSD Method and 95% Confidence

Location*Concentration N Mean Grouping

B 1.0 11 101.898 A M 2.0 10 94.590 A B M 1.0 11 93.284 A B B 0.6 12 89.000 B B 2.0 10 77.840 C M 0.6 12 40.607 D

Means that do not share a letter are significantly different.

Wetland 2 General Linear Model: %DO versus Location, Concentration

Method

Factor coding (-1, 0, +1)

Factor Information

Factor Type Levels Values

Location Fixed 2 B, M Concentration Fixed 3 0.6, 1.0, 2.0

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Location 1 1262 1262.4 8.33 0.006 Concentration 2 8736 4368.2 28.81 0.000 Location*Concentration 2 6687 3343.3 22.05 0.000 Error 46 6974 151.6 Total 51 22856

Model Summary

S R-sq R-sq(adj) R-sq(pred)

12.3129 69.49% 66.17% 61.83%

Regression Equation

%DO = 86.54 + 4.98 Location_B - 4.98 Location_M - 16.15 Concentration_0.6 + 15.82 Concentration_1.0 + 0.33 Concentration_2.0 + 15.60 Location*Concentration_B 0.6 - 2.37 Location*Concentration_B 1.0 - 13.23 Location*Concentration_B 2.0 - 15.60 Location*Concentration_M 0.6 + 2.37 Location*Concentration_M 1.0 + 13.23 Location*Concentration_M 2.0

Fits and Diagnostics for Unusual Observations

Obs %DO Fit Resid Std Resid

5 66.57 90.98 -24.41 -2.12 R 9 158.01 104.97 53.04 4.52 R

R Large residual

Means

Fitted Term Mean SE Mean

Location B 91.52 2.35 M 81.57 2.53 Concentration 0.6 70.39 3.19 1.0 102.37 2.77 2.0 86.87 2.99 Location*Concentration B 0.6 90.98 4.35 B 1.0 104.97 3.71 B 2.0 78.61 4.10 M 0.6 49.81 4.65 M 1.0 99.76 4.10 M 2.0 95.13 4.35 Comparisons for %DO Fisher Pairwise Comparisons: Location

Grouping Information Using Fisher LSD Method and 95% Confidence

Location N Mean Grouping

B 28 91.5190 A M 24 81.5676 B

Means that do not share a letter are significantly different.

Fisher Pairwise Comparisons: Concentration

Grouping Information Using Fisher LSD Method and 95% Confidence

Concentration N Mean Grouping

1.0 20 102.366 A

2.0 17 86.869 B 0.6 15 70.395 C

Means that do not share a letter are significantly different.

Fisher Pairwise Comparisons: Location*Concentration

Grouping Information Using Fisher LSD Method and 95% Confidence

Location*Concentration N Mean Grouping

B 1.0 11 104.971 A M 1.0 9 99.761 A B M 2.0 8 95.128 A B B 0.6 8 90.975 B B 2.0 9 78.611 C M 0.6 7 49.814 D

Means that do not share a letter are significantly different.

Resume MADHURI DINAKAR 315-403-5819 | [email protected] |

OBJECTIVE

To be able to effectively apply the principles of ecological engineering to wastewater treatment with a focus on nutrients, thereby improving water quality and availability of fresh water specifically in rural communities.

SKILLS Technical Skills: Water quality analysis, Design of constructed wetlands and wastewater treatment plants, Microscopy, Biochemical tests, ANOVA Software and Computational Skills: Statistical Software R, Python, ArcGIS, Microsoft Office EDUCATION MSc Ecological Engineering, GPA: 3.97 Aug SUNY College of Environmental Science and Forestry, Syracuse NY 2019 • Thesis: Enhancing ammonia removal in constructed wetlands using hydrogen peroxide i. Achieved mass removal rates of >95% with complete nitrification ii. Enhanced bacterial activity observed, increasing the rate of nitrification • Relevant Courses: Ecological Engineering, Wastewater Engineering, Resources Recovery, Graduate level Statistics, Numerical and Computational Methods • Presented thesis research at 91st Annual NYWEA Conference, New York City • Recipient of O’Brien and Gere Graduate Fellowship B.E Biotechnology, GPA: 3.96 2011-15 PES Institute of Technology, Bengaluru, India • Capstone at Indian Institute of Science: Energy harvesting drone inspired from albatross and hummingbirds - • Relevant Courses: Fluid Mechanics, Microbiology, Heat and Mass Transfer, Biochemistry, Plant Biotechnology EXPERIENCE Graduate Assistant 2016- SUNY College of Environmental Science and Forestry, Syracuse NY Present • Performed water quality research on ammonia removal in treatment wetlands and resources recovery • Educated students in Ecological engineering, Resources Recovery, Statics and Dynamics • Administrative assistant in the Office of Instruction and Graduate Studies Research Assistant Jun- Aug SUNY Research Foundation 2018 • Digitizing data to understand change in different types land-cover over a decade 2008-2018 Project Assistant 2015-16 Indian Institute of Science, Bengaluru India • Developed a tool for rationale capture for the national aerospace industry • Designed a tool, IdeaInspire V.3 for analogical design • Developed a “Bio-translator” tool which could be used to better understand biological systems LEADERSHIP AND RELATED PROJECTS • Secretary at Graduate Student Association Senate at SUNY-ESF 2018-19 • Graduate Student Ambassador, Office of Instruction and Graduate Studies, SUNY-ESF 2018-19 • Review of pharmaceuticals removal in constructed wetlands 2017 • Life Cycle Assessment of Red oxide floors v/s vitrified tiles 2017 • Analyzed the correlation between mosquito transmitted diseases and constructed wetlands location 2016 • Organized several national and international workshops and conferences: ICoRD 2015, ICDC 2015 2013-16 • Conceptualization of woodpecker inspired shock absorbers for motorcycles for the Biomimicry 3.8 Global design 2014 challenge.

PUBLICATIONS Amaresh Chakrabarti, L Siddharth, Madhuri Dinakar, Megha Panda, Neha Palegar, Sonal Keshwani. IDEAINSPIRE 3.0- A tool for Analogical Design, accepted for a podium presentation at the International Conference on Research into Design, Guwahati, 9th -11th January 2017.