Determining Effects of Class I Landfill Leachate on Biological Nutrient Removal in Wastewater Treatment. Dr. Ashley Danley-Thomson

Transcription

1 Determining Effects of Class I Landfill Leachate on Biological Nutrient Removal in Wastewater Treatment February 2018 Dr. Ashley Danley-Thomson Florida Gulf Coast University Environmental and Civil Engineering Hinkley Center for Solid and Hazardous Waste Management University of Florida P. O. Box Gainesville, FL Report #11397

2 PROJECT TITLE: Determining Effects of Class I Landfill Leachate on Biological Nutrient Removal in Wastewater Treatment PRINCIPAL INVESTIGATOR(S): Ashley Danley-Thomson, Ph.D. AFFILIATION: Florida Gulf Coast University CONTACT: (239) , athomson@fgcu.edu PROJECT WEBSITE: PROJECT DURATION: January 2017 January 2018 ABSTRACT: Leachate management challenges at wastewater treatment plants (WWTPs) vary by plant size, percent of leachate to overall flow, leachate composition, waste composition, and regional differences, and are known to include ammonia removal inhibition and biological treatment upset. Both of these issues can be seen in the activated sludge process of the treatment train during biological nutrient removal (BNR) and can be affected by compounds in the leachate matrix. As wastewater treatment operators are increasingly refusing to accept landfill leachate, the overall goal of the research is to determine the effect of landfill leachate on nitrifying activated sludge activity and determine if activated sludge can be adapted to handle leachate loadings that are known to cause overloading. The effect of several landfill leachate sources on nitrifying activated sludge activity was quantified using Specific Oxygen Uptake Rate (SOUR). Additionally, the effect of leachate on the efficacy of BNR activated sludge processes using lab scale sequencing batch reactors (SBRs) was determined. Each reactor received a leachate loading of 5%, 10%, 15%, or 20% of total flow for three solid retention times (SRTs). Controls were also operated (100% loading of leachate as positive control, 100% loading of activated sludge as negative control). To determine if the sludge could be adapted to a leachate loading known to cause overloading, each reactor was charged with a loading of 30% leachate for 7 days. Results showed that SBRs previously adapted to leachate loadings of up to 20% v/v had improved COD removal when charged with an organic overloading of 30% v/v when compared to SBRs without previous exposure to leachate. The SBRs adapted to a 5% leachate loading exhibited greater amounts of ammonium removal and TIN-N removal than the control when exposed to the 30% v/v loading. SBRs exposed to 10% leachate loading exhibited similar ammonium removal rates to the control but had less nitrite accumulation when exposed to the 30% v/v loading. Results indicate that BNR activated sludge may be adapted to handle higher loads of landfill leachate in terms of COD removal and TIN removal than BNR activated sludge that has never been exposed to leachate. Key words: Ammonium (NH4 + -N); landfill leachate; activated sludge; nitrification; activated sludge; inhibition. ii

3 METRICS REPORTING Student Researchers: Name: Alexander Brawley Anticipated Degree: B.S. Environmental Engineering Department: Environmental and Civil Engineering Name: Sandra Un Jan Anticipated Degree: B.S. Environmental Engineering Department: Environmental and Civil Engineering Name: Shane Herman Anticipated Degree: B.S. Environmental Engineering Department: Environmental and Civil Engineering Name: Kendall Karcher Anticipated Degree: B.S. Environmental Engineering Department: Environmental and Civil Engineering Name: Morgan Humphrey Anticipated Degree: B.S. Environmental Engineering Department: Environmental and Civil Engineering Name: Lucas Parmer Anticipated Degree: B.S. Environmental Engineering Department: Environmental and Civil Engineering Name: Connor Haydon Anticipated Degree: B.S. Environmental Engineering Department: Environmental and Civil Engineering Name: Austin Wise Anticipated Degree: B.S. Environmental Engineering Department: Environmental and Civil Engineering Metrics: iii

4 1. List research publications resulting from THIS Hinkley Center project. None at this time. 2. List research presentations resulting from THIS Hinkley Center project. Oral Presentations: National Academy of Engineering Grand Challenges & Undergraduate Research Symposium on providing access to clean water and managing the nutrient cycle, September 6, 2017 and October 25, 2017, Fort Myers, FL. Global Development Engineering. FGCU Undergraduate Research Symposium, December 8, 2017, Fort Myers, FL. Determining Effects of Class I Landfill Leachate on Biological Nutrient Removal in Wastewater Treatment. Florida Collegiate Honors Council Conference, February 9-11, 2018, Fort Myers, FL. Determining effects of leachate-associated toxicity and dissolved organic nitrogen on biological wastewater treatment and effluent-receiving waters. Poster Presentations: TAG Meeting September 13, 2016 FGCU Research Day 2018, April 18, 2017, Fort Myers, FL Solid Waste Association of North America, Florida Section Annual Conference and Hinkley Center Colloquium, July 23 25, 2017, Sanibel, Florida. Determining Effects of Class I Landfill Leachate on Biological Nutrient Removal in Wastewater Treatment. TAG Meeting October 18, 2017 TAG Meeting January 24, th Annual Southwest Florida Water Resources Conference, American Water Resources Association. Student Water Resources Research Poster Contest, February 2, 2018, Fort Myers, FL. Determining Effects of Class I Landfill Leachate on Biological Nutrient Removal in Wastewater Treatment. Winner of Undergraduate Research Poster Presentation. Florida Collegiate Honors Council Conference, February 9-11, 2018, Fort Myers, FL. Determining effects of leachate-associated toxicity and dissolved organic nitrogen on biological wastewater treatment and effluent-receiving waters. FGCU Research Day 2018, April 17, 2018, Fort Myers, FL. 3. List who has referenced or cited your publications from this project. None at this time. 4. How have the research results from THIS Hinkley Center project been leveraged to secure additional research funding? What grant applications have you submitted or are planning on submitting? Grant application submitted to EREF, but was not accepted for funding. iv

5 Additional research funding was secured to hire an additional student over the summer to work on this project. This student will lead the effort for Objective I (leachate collection and SOUR tests) over the next few months. Research plan and findings so far were submitted to an internal FGCU grant from the Blair Foundation ($5,000; June 2017 August 2017). Several additional grants have been submitted related to this work and nutrient management 5. What new collaborations were initiated based on THIS Hinkley Center project? FGCU is now working with several consulting firms and municipalities with whom FGCU did not previously have contact. Specifically, Collier County Solid Waste, Lee County Solid Waste, Heart of Florida Landfill, Waste Management and the City of Ocala. 6. How have the results from THIS Hinkley Center funded project been used (not will be used) by the FDEP or other stakeholders? (1 paragraph maximum). Freely describe how the findings and implications from your project have been used to advance and improve solid waste management practices. This research provides valuable information on how activated sludge in wastewater treatment plants can be adapted to handle leachate loads. This information has not yet been used by the FDEP or stakeholders, but we have been told that it provides valuable information, so we are hopeful it will be used in the future. ACKNOWLEDGEMENTS v

6 This work is funded by the Hinkley Center for Solid and Hazardous Waste. Special thanks to our Technical Awareness Group who have helped developed the direction of this research and provided feedback on various aspects of this project. Thank you to John Langan for his guidance and laboratory support, and the numerous Landfill and WWTP operators who have offered their support. Student Researchers Name: Alexander Brawley Department: Environmental and Civil Engineering Bio: Alex is a senior Environmental Engineering student. After graduating in May 2018, he will continue working in sustainable development at Suffolk Construction. Name: Sandra Un Jan spunjan5318@eagle.fgcu.edu Department: Environmental and Civil Engineering Bio: Sandra is a junior Environmental Engineering student. Sandra aspires to attend graduate school, earn her Ph.D. and work in international development and water quality issues. Name: Shane Herman stherman7754@eagle.fgcu.edu Department: Environmental and Civil Engineering Bio: Shane is a senior Environmental Engineering student. After graduation in May 2018, Shane aspires to attend graduate school to obtain a Masters degree and possibly a Ph.D. Name: Kendall Karcher kekarcher7336@eagle.fgcu.edu Department: Environmental and Civil Engineering Bio: Kendall is an honors sophomore Environmental Engineering student. Kendall aspires to obtain a Ph.D. and work on water quality issues in the Indian River Lagoon on the East coast of Florida. Name: Morgan Humphrey mehumphrey6804@eagle.fgcu.edu Department: Environmental and Civil Engineering Bio: Morgan is an honors sophomore Environmental and Civil Engineering student. Morgan aspires to work in international development on increasing capacity and improving infrastructure in Central and South America to help countries improve their outcomes to severe weather events such as El Nino. Name: Lucas Parmer lpparmer1805@eagle.fgcu.edu Department: Environmental and Civil Engineering Bio: Lucas is a senior Environmental Engineering student. After graduation in May 2018, Lucas aspires to work in private industry as a consulting engineer. vi

7 Name: Connor Haydon Department: Environmental and Civil Engineering Bio: Connor graduated with a Bachelors of Science in Environmental Engineering in May of Connor now works in the field of Solid Waste Engineering as a Landfill Manager in Michigan. Name: Austin Wise amwise9467@eagle.fgcu.edu Department: Environmental and Civil Engineering Bio: Austin in a sophomore Environmental and Civil Engineering student. Austin is the President of Engineering Community Outreach, an international service-based student organization at FGCU. Austin aspires to continue working in International Development upon graduation. Technical Awareness Group (TAG) members Wester W. Henderson III, Research Coordinator III, Hinkley Center for Solid and Hazardous Waste Management, Gainesville, FL Debra Reinhart, PhD, PE, BCEE, Professor and Assistant Vice President for Research, University of Central Florida, Orlando, FL. Kelly Campbell, Landfill Manager, Collier County Solid Waste, Naples, FL. Ronald Cavalieri, PE, BCEE, Principal Engineer/Associate Vice President, AECOM, Fort Myers, FL. Patty DiPiero, Legislative and Compliance Programs Manager, Lee County Utilities, Fort Myers, FL. Isaac Holowell, Engineer II, CDM Smith, Fort Myers, FL. Ryan Holzem, PhD, ENV SP, EIT, Assistant Professor, University of Wisconsin, Green Bay, WI. Thomas Worley-Morse, PhD, PE, Principal Scientist, Hazen and Sawyer, Raleigh, NC. Stephanie Bolyard, PhD, Research and Scholarship Program Manager, Environmental Research and Education Foundation (EREF), Raleigh, NC. Amir Motlagh, PhD, PhD Candidate, University of Central Florida, Orlando, FL. Christina Alito, PhD, PE, ENV SP, Water/Wastewater Engineer, HDR Engineering, Vienna, VA. Rebecca Rodriguez, P.E., Engineering Manager II, Lee County Solid Waste Division, Fort Myers, FL. Linda Monroy, P.E., Project Manager Associate, Lee County Solid Waste Division, Fort Myers, FL. vii

8 Table of Contents Lists of Tables... ix Lists of Figures... x List of Acronyms and Abbreviations... xii Executive Summary... xiii Introduction... 1 Methods... 3 Results... 9 Conclusions Bibliography viii

9 List of Tables Table 1. Landfill characteristics... 6 Table 2. Average loading for each condition in Phase I Table 3. Average effluent for each condition at the end of Phase I Table 4. Average loading for each condition in Phase II Table 5. Average effluent for each condition at the end of Phase II Table 6. Average loading for each adapted sludge SBR condition in Phase III Table 7. Average effluent for each adapted sludge SBR condition in Phase III Table 8. SOUR Index of each leachate loading condition simulated in Phase II during Phase III when each SBR received a 30% leachate loading Table 9. Leachate characteristics and SOUR Index of leachate samples ix

10 List of Figures Figure 1. Photo of reactor setup. Each reactor contains nitrifying activated sludge and is fed with synthetic wastewater Figure 2. SBRs are operated with timers, peristaltic pumps, stir plates and aerators Figure 3. Layout of reactor conditions in Phase II. Six conditions are operated in duplicate Figure 4. Setup of SOUR test... 8 Figure 5. Total inorganic nitrogen distribution in effluent of each SBR leachate loading condition. Bars show relative percentage of each inorganic nitrogen species in the effluent, as well as any TIN removal Figure 6. COD in effluent of each SBR leachate loading condition. Bars show relative percentage of COD in the effluent, as well as any COD removal. Each stacked bar is labeled with COD concentration in the effluent and COD removal in mg/l Figure 7. TIN-N in the effluent of each SBR based on leachate loading condition they were adapted to. Bars show relative percentage of each inorganic nitrogen species in the effluent, as well as any TIN removal Figure 8. COD in effluent of each SBR with the leachate loading condition that each reactor was adapted to. Bars show relative percentage of COD in the effluent, as well as any COD removal Figure 9. Removal of TIN-N per mg of COD loading in Phase III for each leachate loading condition Figure 10. SOUR index and leachate loading each SBR is adapted to over the 7 days of leachate loading at 30% x

11 Figure 11. SOUR index of each leachate sample tested for Objective xi

12 List of Acronyms and Abbreviations AOB BNR BOD COD DO EDTA EPA FGCU HRT NOB POTW SBR SOUR SRT TIN TN TSS VSS WWTP Ammonia-oxidizing bacteria Biological nutrient removal Biochemical oxygen demand Chemical oxygen demand Dissolved oxygen Ethylenediamine tetraacetic acid Environmental Protection Agency Florida Gulf Coast University Hydraulic retention time Nitrate-oxidizing bacteria Publicly operated treatment works Sequencing batch reactor Specific oxygen uptake rate Solids retention time Total inorganic nitrogen Total nitrogen Total suspended solids Volatile suspended solids Wastewater treatment plant xii

13 Executive Summary Leachate management challenges at wastewater treatment plants (WWTPs) vary by plant size, percent of leachate to overall flow, leachate composition, waste composition, and regional differences, and are known to include ammonia removal inhibition and biological treatment upset. Both of these issues can be seen in the activated sludge process of the treatment train during biological nutrient removal (BNR) and can be affected by compounds in the leachate matrix. As wastewater treatment operators are increasingly refusing to accept landfill leachate, the overall goal of the research is to determine the effect of landfill leachate on nitrifying activated sludge activity and determine if activated sludge can be adapted to handle leachate loadings that are known to cause overloading. The effect of several landfill leachate sources on nitrifying activated sludge activity was quantified using Specific Oxygen Uptake Rate (SOUR). Additionally, the effect of leachate on the efficacy of BNR activated sludge processes using lab scale sequencing batch reactors (SBRs) was determined. Each reactor received a leachate loading of 5%, 10%, 15%, or 20% of total flow for three solid retention times (SRTs). Controls were also operated (100% loading of leachate as positive control, 100% loading of activated sludge as negative control). To determine if the sludge could be adapted to a leachate loading known to cause overloading, each reactor was charged with a loading of 30% leachate for 7 days. Therefore, this study aimed to address the following objectives: Objective 1: Quantify the effect of various landfill leachate sources on nitrifying activated sludge utilizing Specific Oxygen Uptake Rate (SOUR). Six landfill leachate samples were tested at discrete ratios to determine how leachate loading may affect sludge activity. Objective 2: Evaluate the effect of leachate on the efficacy of biological nutrient removal (BNR) activated sludge processing using lab scale sequencing batch reactors (SBRs). SBRs were operated with nitrifying activated sludge and fed distinct ratios of synthetic wastewater and landfill leachate. Controls were also operated (100% loading of leachate as positive control, 100% loading of activated sludge as negative control). Objective 3: Determine the extent that BNR activated sludge can be adapted to effectively handle a loading of landfill leachate known to cause overloading using lab scale sequencing batch reactors (SBRs). SBRs operated with nitrifying activated sludge adapted to leachate loadings as described in Objective 2 were fed a ratio of 70% synthetic wastewater and 30% landfill leachate for one week. Results showed that SBRs previously adapted to leachate loadings of up to 20% v/v had improved COD removal when charged with an organic overloading of 30% v/v when compared to SBRs without previous exposure to leachate. The SBRs adapted to a 5% leachate loading exhibited greater amounts of ammonium removal and TIN-N removal than the control when exposed to the 30% v/v loading. SBRs exposed to 10% leachate loading exhibited similar ammonium removal rates to the control but had less nitrite accumulation when exposed to the 30% v/v loading. Results indicate that BNR activated sludge may be adapted to handle higher loads of landfill leachate in terms of COD removal and TIN removal than BNR activated sludge that has never been exposed to leachate. xiii

14 Introduction When wastewater treatment facilities receive loads from significant industrial waste streams, effluent quality can vary due to the complexity of the waste stream. Leachate, specifically, is known to be complex, potentially containing constituents such as heavy metals, dissolved organic matter, inorganic macro components, xenobiotic organic matter, microbes, hexachlorobutadiene, pentachlorobenzene, PAHs, detergents, volatile organic compounds, organophosphates, oils, and grease (Dalzell et al, 2002; Zhang et al, 2016; Matejczyk et al, 2011). Biological treatment is vulnerable to discharges of industrial wastewater and any adverse effect on this stage of treatment can reduce the quality of treated effluent that is discharged to the receiving body of water (Dalzell et al, 2002). Specifically, the effect of landfill leachate on the biological secondary treatment process, which is mediated by environmentally-sensitive organisms, is known to have implications on the overall efficacy of the wastewater treatment process at certain loadings, but studies have generally concluded that leachate does not adversely affect WWTP removal efficiencies at loadings of 10% or less in laboratory settings (Diamadopoulos et al., 1997; Çeçen & Aktaş, 2004; Çeçen & Çakıroğlu, 2001), although inhibition at leachate loadings of more than 4% have been observed in operational wastewater treatment plants (Brennan et al., 2017), indicating that hydraulic-loading based acceptance criteria recommendations may not be appropriate when cotreating leachate unless leachate ammonium and COD composition is considered. The main goals of the BNR activated sludge process in wastewater treatment are to 1) decrease biological oxygen demand by ~90%, and 2) promote the process of nitrification, degrading ammonium into nitrate, and denitrification, the degradation of nitrate into nitrogen gas. Ammoniaoxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) facilitate the process of nitrification, but are known to be environmentally-sensitive organisms. Water quality fluctuations outside of a relatively small range can cause inhibitions in the AOB and NOB populations, and therefore inhibit the treatment process (Wisniowski et al, 2005). Concerns exist regarding the effects of leachate loading to AOB and NOB, which could reduce the efficacy of the biological treatment process, and the overall facility. This has been observed to occur when oxygen supply is not sufficient due to overloading of COD, when the ph of the mixed liquor falls outside of the optimal range for AOB and NOB, and when ammonia toxicity is present (Wisniowski et al, 2005; Brennan et al., 2017; Çeçen & Çakıroğlu, 2001). The effectiveness of biological nutrient removal depends on the ability of nitrifying organisms to oxidize ammonia to nitrate. This process requires oxygen and when influents with varying concentrations of COD are input into activated sludge systems, optimal efficiency can be compromised due to overloading (Surmacz-Gorska et al, 1996). In nitrification, ammonia-oxidation is usually the rate limiting step, so nitrite does not typically accumulate during the nitrification process (Henze et al., 1997). However, nitrite accumulation has been seen during co-treatment of landfill leachate during nitrification (Çeçen & Çakıroğlu, 2001; Hwang et al., 2000; Liu & Capdeville, 1994) and denitrification (Hongwei et al., 2009). The occurrence of inhibition by free ammonia on ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) is well known (Anthonisen et al., 1976; Turk et al, 1989; Kim et al., 2006). NOB are known to be inhibited by NH3-N in the range of mg/l while AOB are inhibited in the range of mg/l (Welander et al, 1998), and therefore nitrite build-up can be seen at elevated free ammonia levels. Other possible inhibitions to BNR include heavy metals and xenobiotic organic matter that may be present in leachate. 1

15 Therefore, this study aimed to address the following objectives: Objective 1: Quantify the effect of various landfill leachate sources on nitrifying activated sludge utilizing Specific Oxygen Uptake Rate (SOUR). Six landfill leachate samples were tested at discrete ratios to determine how leachate loading may affect sludge activity. Objective 2: Evaluate the effect of leachate on the efficacy of biological nutrient removal (BNR) activated sludge processing using lab scale sequencing batch reactors (SBRs). SBRs were operated with nitrifying activated sludge and fed distinct ratios of synthetic wastewater and landfill leachate. Controls were also operated (100% loading of leachate as positive control, 100% loading of activated sludge as negative control). Objective 3: Determine the extent that BNR activated sludge can be adapted to effectively handle a loading of landfill leachate known to cause overloading using lab scale sequencing batch reactors (SBRs). SBRs operated with nitrifying activated sludge adapted to leachate loadings as described in Objective 2 were fed a ratio of 70% synthetic wastewater and 30% landfill leachate for one week. As wastewater treatment operators are increasingly hesitant accept landfill leachate, this study will gauge the specific impacts of leachate on the biological wastewater treatment and provide data that will allow WWTPs to decrease biological treatment disruptions and ammonia removal inhibition that occur as a result of leachate management. These leachate management challenges are critical, as the development of the Numeric Nutrient Criteria for Florida s waters affect the effluent limits for WWTPs. 2

16 Methods SBR Design. The construction, operation, and leachate loading tests are conducted based on previously published work (Arnaout et al., 2014). Twelve 8-L bench scale SBRs are constructed out of square pieces of plexiglass (8 x 8 x 8 in, 1/8 in. thick) and welded together using poly(methyl methacrylate) (Figure 1 3). The peak volume achieved in all reactors was 3.0 L and the volume after decanting was approximately 2.0 L. Throughout the study (Phase I III), SBRs were operated on an 8 h cycle, starting with i) 3 min of influent synthetic wastewater and leachate feeding, ii) 3 h of aeration and vigorous mixing, iii) 3.3 h of mixing only, iv) 1 h of settling, v) 3 min of decanting, and vi) 30 min of idle. A solid retention time (SRT) of approximately 14 d is utilized to ensure optimal conditions for nitrification and a hydraulic retention time (HRT) of 24 h is used. The SRT was maintained by wasting sludge periodically to retain 4,000 5,000 mg/l of mixed liquor suspended solids (MLSS). All reactors were covered with aluminum foil to prevent any photolysis. Figure 1. Photo of reactor setup. Each reactor contains nitrifying activated sludge and is fed with synthetic wastewater. 3

17 Figure 2. SBRs are operated with timers, peristaltic pumps, stir plates and aerators. Phase I. All reactors receive synthetic wastewater. Activated sludge was obtained from a local advanced wastewater treatment facility (Fort Myers, FL) which currently treats 3 MGD and completes biological nutrient removal of biochemical oxygen demand (BOD), NH4 +, and phosphorus. Approximately 3 L of activated sludge inoculum was used to seed the each of the ten SBRs with an active microbial community. During inoculation, the activated sludge was added directly to each empty SBR and the first 8 h SBR cycle occurred. Following the first 8 h cycle, 1 L of activated sludge was wasted and the SBRs were charged with 1 L of simulated wastewater based on a recipe developed by Zeng et al (2003) and previously used in the authors laboratory (Alito and Gunsch, 2014; Danley-Thomson et al, 2015). The synthetic wastewater had an average COD of 599 mg/l, average ammonia concentration of 66 mg/l, and ph of 7. Two L of the simulated wastewater consisted of 100 ml of solution A and 1.9 L of solution B. Solution A contained g/l NaCH3CO - 2,1 g/l MgSO4, 0.45 g/l CaCl2, 3.3 g/l NH4Cl, 0.5 g/l peptone and 6 ml/l of a nutrient solution (recipe provided below). Solution A was adjusted to ph 5.5 with 2 M HCl and autoclaved. Solution B contained 28.5 mg/l KH2PO4 and 32.5 mg/l K2HPO4 and was adjusted to ph 10 with 2 M NaOH. The nutrient solution consisted of 1.5 g/l FeCl3, 0.15 g/l H3BO3, 0.03 g/l CuSO4, 0.18 g/l KI, 0.12 g/l MnCl2, 0.06 g/l Na2MoO4, 0.12 g/l, ZnSO4, 0.15 g/l CoCl2, and 10 g/l ethylenediamine tetraacetic acid (EDTA). All chemicals were obtained from Sigma Aldrich. The simulated wastewater was prepared every other day and added to the SBRs using a metered pump (Masterflex, Cole-Parmer, Vernon Hills, IL). Ten SBRs (1A 5B) are fed this synthetic wastewater in Phase I until steady state COD and nitrogen degradation was reached (three SRTs). Reactors 6A and 6B are operated using only 4

18 landfill leachate for the duration of the study (Phase I Phase III) in order to serve as the positive control. Phase II. Reactors receive defined ratios of synthetic wastewater and landfill leachate until steady state is achieved. After steady state COD and nitrogen removal was achieved in Phase I, the experiment entered Phase II where leachate was mixed with synthetic wastewater at distinct ratios and charged to the reactors in duplicate (see Figure 3). To adapt sludge to leachate loadings during Phase II, reactors 1A and 1B were the negative control receiving only synthetic wastewater and no leachate (0% v/v), reactor 2A and 2B receive a continuous supply of 5% mixture v/v of leachate and synthetic wastewater, 3A and 3B receive a continuous supply of 10% mixture v/v of leachate and synthetic wastewater, 4A and 4B receive a continuous supply of 15% mixture v/v of leachate and synthetic wastewater, and 5A and 5B receive a continuous supply of 20% mixture v/v of leachate and synthetic wastewater. Reactor 6A and 6B were the positive controls and received only leachate (100% v/v). Reactors were operated in this fashion for three SRTs (6 weeks), until steady state N and COD removal was achieved. Figure 3. Layout of reactor conditions in Phase II. Six conditions are operated in duplicate. Phase III. All reactors receive an organic over-loading of landfill leachate (30% v/v). After steady state was achieved, all reactors were charged with a 30% leachate v/v loading for one week to determine COD and nitrogen removal (Phase III), which corresponded to an organic loading rate of 3.6 g COD / (L d). 5

19 Analytical Methods. To monitor COD and N removal efficiency, influent and effluent samples were collected approximately every 7 d. Five parameters were measured using HACH reagents (Loveland, CO): COD (mercuric digestion method), ammonia (salicylate method), nitrite (diazotization method), nitrate (cadmium reduction method), and phosphate (ascorbic acid method). Samples were taken directly from SBR containers immediately before timing period v, three min of decanting. Total suspended solids (TSS) and volatile suspended solids (VSS) were measured according to standard methods as described in The Standard Methods for the Examination of Water and Wastewater (2005). BOD was measured in 300 ml glass BOD bottles using standard methods (EPA, Method 5210 B); approximately 0.16 g of Hach nitrification inhibitor was added to each 300 ml bottle (Loveland, CO), followed by Hach BOD nutrient buffer pillows (Loveland, CO). Measurements were recorded in duplicate. Dissolved oxygen (DO) and ph measurements were also obtained for each SBRs influent and effluent. Specific Oxygen Uptake Rate (SOUR). In order to evaluate how landfill leachate may affect the activity of BNR activated sludge, SOUR was conducted for landfill leachate samples (Table 1). Six landfill sites in southwest and central Florida were sampled over a 6-month sampling period, including the leachate charged to the SBRs in Phase II and III. The sites sampled represented a variety of factors including rural and urban areas, industrial activity, and capped sites and those sites currently accepting waste. Sample Site Table 1. Landfill characteristics MSW Collected (Ton/year) a Landfill Type 1 NA Rural 2 229,617 Rural 3 108,227 Regional 4 b 20,312 Regional 5 244,125 Regional 6 0 Semiurban/capped a Data from 2017 b Leachate utilized in Phase II and III SOUR was measured according to EPA Method 1683 (EPA, 2001). Briefly, fresh activated sludge was collected from the aeration stage of a biological nutrient removal activated sludge tank from a BNR wastewater treatment facility in Fort Myers, Florida (same facility where the activated sludge was retrieved for the SBRs). Activated sludge was transported to Florida Gulf Coast University (FGCU) and kept under aeration. Within one hour of collection, 300 ml BOD bottles were filled with mixtures of landfill leachate and fresh activated sludge (0%, 1%, 5%, 10%, 20%, 6

20 50%, and 75% mixture of leachate and activated sludge v/v). Biomass was oxygenated prior to addition to the BOD bottles to ensure that DO in the reaction vessel was greater than 2 mg/l. For the endogenous control (0% leachate v/v), the liquid level in the reactor was adjusted to ensure that there was no headspace (see Figure 4). The DO probe was inserted and plumbers putty was used to seal the reactor to avoid oxygen penetration from the atmosphere. Oxygen concentration was monitored for 30 minutes and recorded every 30 sec. The first derivative (slope) was utilized as the endogenous oxygen uptake rate. For the experimental vessels, landfill leachate was added at the aforementioned ratios. As with the endogenous control, the liquid level was adjusted so there was no headspace, the DO probe inserted, and plumbers putty utilized to seal the bottle. DO was monitored and recorded for 30 min. TSS and VSS of each mixture was measured according to standard methods. Specific oxygen uptake rate was calculated by dividing the oxygen uptake rate (mg/l/s) by the biomass concentration (VSS) (g/l). Specific oxygen uptake rate (SOUR) was used as an indicator for how leachate additions may affect the activity of BNR activated sludge. SOUR is typically used to understand if a particular sludge is overloaded with COD, is experiencing a toxic response to a particular waste, is underloaded and needs to experience sludge wasting, or to know that the organic loading and VSS are well-balanced (Gutierrez et al, 2002; Surmacz-Gorska et al, 1996). For the purposes of this experiment, a SOUR index was utilized where the control (0% leachate loading) defined as a SOUR index of one, numbers above one indicate more oxygen uptake rate per g of VSS than the control, which indicates increased activity of the microbes and possible organic overloading, and a SOUR index of less than one indicates less oxygen uptake rate per g of VSS than the control, which could indicate organic underloading and a need for sludge wasting, or it could indicate that the microbes are experiencing a toxic response and therefore their activity is decreased. When comparing leachate samples from different landfills, differences in SOUR index values may suggest differences in biodegradability of the leachate samples. If leachate is composed of higher amounts of non-biodegradable carbon, the oxygen uptake rate will be lower than a leachate with higher amounts of biodegradable carbon. SOUR tests were conducted on the MLSS of each SBR during the 7 days of Phase III where each SBR received a loading of 30% leachate. The SOUR Index gives an indication of how each SBR microbial community was affected by the 30% leachate loading and if adapting the sludge in Phase II had an affect on the community s activity in Phase III. 7

21 Figure 4. Setup of SOUR test Statistical Analysis. The unpaired, two tailed student s t-test was used to identify statistical differences between control samples and treated samples for all data analysis 8

22 Results Phase I. Reactor Start Up For three SRTs (6 weeks), ten SBRs were operated with BNR activated sludge adapted to synthetic wastewater. Table 2 provides a water quality summary for the wastewater used in this study. Effluent characteristics are shown in Table 3. Briefly, at an influent organic loading rate of 0.60 g COD/(L d), the average effluent concentrations of ammonium, nitrite, nitrate, COD, and phosphate were mg/l, mg/l, mg/l, mg/l, and mg/l, respectively. This represented COD, TIN, and TP removals of 91.0%, 69.4%, and 35.8%, respectively. The average effluent COD/TIN ratio was 2.5. TIN removal averaged mg/l. The average VSS in all 10 reactors receiving synthetic wastewater was 4, mg/l. The average VSS in the SBRs receiving only leachate was mg/l. No statistically significant difference in treatment was observed in the SBRs receiving synthetic wastewater (p>0.05). No significant nitrogen removal was observed for the SBRs receiving only landfill leachate (p>0.05). Table 2. Average loading for each condition in Phase I. Reactor Leachate Loading (% v/v) Influent COD (mg/l) Influent TIN- N (mg/l) Influent COD/TIN- N ratio (mg/mg) Influent Organic Loading Rate (g COD / (L d)) g COD in influent / g VSS in SBR g TIN-N in influent / g VSS in SBR , ,

23 Table 3. Average effluent for each condition at the end of Phase I. Reactor Leachate Loading (% v/v) Effluent COD (mg/l) Effluent TIN-N (mg/l) Effluent COD/TIN-N ratio (mg/mg) COD Removal (%) COD Removal (mg) TIN-N Removal (%) TIN-N Removal (mg) , , Phase II. Leachate Conditioning. Characteristics of the leachate sample throughout the experiment were COD = 5, ,222.8 mg/l, NH4 + = 2, mg/l, NO3 - = mg/l, NO2 - = mg/l, PO4 - = mg/l, and ph of The average daily leachate loading accounted for 39.1, 55.4, 70.5, and 75.2% of the COD loading and 70.3, 80.1, 90.1, and 91.9% of TIN loadings to SBRs for the 5, 10, 15, and 20% loadings, respectively. All reactors were loaded with a relatively high C/N ratio as indicated by the COD/TIN-N in Table 4. Low C/N values can inhibit biological processing, but these values indicate satisfactory levels of carbon for dentification of nitrite and nitrate (Wu et al., 2015). Figure 5 shows total inorganic nitrogen distribution in the effluents of each SBR leachate loading condition. Generally, as the leachate loading rate increased, TIN removal decreased, possibly due to the inhibition of nitrification by the oxygen demand of the organic compounds found in the leachate (Im et al, 2001). The reactor receiving only synthetic wastewater (0% leachate loading) had a 76.9% TIN-N removal rate, and 92.6% ammonium removal. The SBRs receiving 5% leachate loading had a 81.4% TIN-N removal rate, and a 98.5% ammonium removal. The SBRs receiving 10% leachate loading had a 69.4% TIN-N removal rate and a 84.0% ammonium removal. These observations suggest that nitrification was not inhibited at leachate loadings of 5% and 10%, which is in agreement with previous studies (Diamadopoulos et al., 1997; Çeçen & Aktaş, 2004; Çeçen & Çakıroğlu, 2001). Nitrate concentrations in the effluent of 5% and 10% were higher than the control which may indicate inhibition to the denitrification process, possibly due to the need for a supplementary carbon source (Ilies & Mavinic, 2001; Bae et al, 1997), as the remaining COD may have been non-biodegradable COD. However, it was observed that inorganic nitrogen conversion percent removal was affected at leachate loadings above 15%, as has been seen in previous studies (Diamadopoulos et al., 1997; Çeçen & Aktaş, 2004; Çeçen & Çakıroğlu, 2001). For leachate loadings of 15% and 20%, TIN-N removal was 35.7% and 28.7% respectively, and ammonium removal was 50.2% and 52.8%, respectively. Additionally, nitrite build up was observed for leachate volumetric loading rates of 10

24 Total Inorganic Nitrogen (mg/l - N) 15% and 20%, indicating that nitritation occurred but nitratation was limited. This may be due to NOB inhibition, possibly due to free ammonia levels. Also, it has been observed that the typical inhibition threshold for ammonium is 480 mg/l for nitrifying populations (Gerardi, 2002; Henze et al, 2002). It can be seen that the 15% and 20% loaded SBRs had influents above this threshold (Table 4). It is interesting to note that although over percent removal of TIN decreased with increasing leachate loading, the actual amount of TIN (mg) increased as the leachate loading increased. This observation may suggest that the actual TIN removal may be more a limitation of the system and operation parameters than of the leachate quality. The SBRs receiving 15% leachate v/v had a COD per VSS loading of 0.39 (Table 4), which is higher than anticipated, and experienced lower rates of nitrification. This is likely due to relatively low concentrations of VSS near the end of the phase (4208 mg/l as opposed to the average of 5,305 mg/l for the other treatment conditions). Nitrate concentrations in the effluent of 15% and 20% were higher than the control and lower leachate loading rates, which may indicate lower denitrification rates. The average COD and TIN loading for each condition is shown in Table 4. Inorganic Nitrogen Distribution - End Phase II 100% % % % % 50% % 30% 20% 10% 0% , % 5% 10% 15% 20% 100% Leachate Volumetric Loading Rate (%) Ammonium Nitrite Nitrate Nitrogen Removed Figure 5. Total inorganic nitrogen distribution in effluent of each SBR leachate loading condition. Bars show relative percentage of each inorganic nitrogen species in the effluent, as well as any TIN removal. 11

25 Table 4. Average loading for each condition in Phase II. Reactor Leachate Loading (% v/v) Influent COD (mg/l) Influent TIN- N (mg/l) Influent COD/TIN- N ratio (mg/mg) Influent Organic Loading Rate (g COD / (L d)) g COD in influent / g VSS in SBR g TIN-N in influent / g VSS in SBR , , , , , , The organic removal rates at the end of Phase II were 0.51, 0.64, 0.64, 0.69, and 0.54 g COD / (L d) for the SBRs conditioned to 0, 5, 10, 15, and 20% sludge, respectively. It can be seen that mg of COD removal generally increased as the leachate loading rate increased (Table 5, Figure 6), but overall COD removal rates (%) decreased, as has been observed in previous studies (Neczaj et al, 2008; Çeçen & Aktaş, 2004), where effluent COD increases as leachate loading increases, due to the increasing load of COD for each leachate loading condition. 12

26 COD (mg/l) COD Distribution - End Phase II 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 1, , , % 5% 10% 15% 20% 100% Leachate Volumetric Loading Rate (%) Effluent COD COD Removed Figure 6. COD in effluent of each SBR leachate loading condition. Bars show relative percentage of COD in the effluent, as well as any COD removal. Each stacked bar is labeled with COD concentration in the effluent and COD removal in mg/l. Table 5. Average effluent for each condition at the end of Phase II. Reactor Leachate Loading (% v/v) Effluent COD (mg/l) Effluent TIN-N (mg/l) Effluent COD/TIN- N ratio (mg/mg) COD Removal (%) COD Removal (mg) TIN-N Removal (%) TIN-N Removal (mg) , , ,

27 Phase III. Leachate Challenge. In Phase III, the influent organic loading rate was increased to 3.6 g COD / (L d), which was double for the reactors adapted to 20% leachate loading (Phase II organic loading rate of 1.8, see Table 4), and represented a greater than 6X increase for the reactor adapted only to synthetic wastewater and not leachate (Phase II organic loading rate of 0.6, see Table 4). The average daily leachate loading accounted for 80.7% of the COD loading to the SBRs and 80.1% of TIN loadings to SBRs (Table 6). Table 6. Average loading for each adapted sludge SBR condition in Phase III. Reactor Adapted to Leachate Loading (% v/v) Leachate Loading (% v/v) Influent COD (mg/l) Influent TIN-N (mg/l) Influent COD/TIN- N ratio (mg/mg) Influent Organic Loading Rate (g COD / (L d)) g COD in influent / g VSS in SBR g TIN-N in influent / g VSS in SBR , , , ,

28 Table 7. Average effluent for each adapted sludge SBR condition in Phase III. Reactor Adapted to Leachate Loading (% v/v) 0 Leachate Loading (% v/v) Effluent COD (mg/l) 2, , , , , , , Effluent TIN-N (mg/l) , Effluent COD/TIN- N ratio (mg/mg) COD Removal (%) COD Removal (mg) TIN-N Removal (%) TIN-N Removal (mg) It can be seen that the SBRs adapted to a leachate loading rate of 5% and 10% experienced increased TIN-N removal than the SBRs adapted to no leachate loading (Table 7). This is also seen using the mg TIN-N removal per mg of COD loading metric (Figure 10). 15

29 Total Inorganic Nitrogen (mg/l - N) Phase III Inorganic Nitrogen Distribution , % 5% 10% 15% 20% 100% Leachate Volumetric Loading Rate (%) Ammonium Nitrite Nitrate Nitrogen Removed Figure 7. TIN-N in the effluent of each SBR based on leachate loading condition they were adapted to. Bars show relative percentage of each inorganic nitrogen species in the effluent, as well as any TIN removal. Figure 7 shows TIN-N removal for each SBR during Phase III. The SBRs adapted to a 5% leachate loading exhibited greater amounts of ammonium removal and overall TIN-N removal. SBRs exposed to 10% leachate loading exhibited similar ammonium removal rates but had less nitrite accumulation. The SBRs adapted to 20% leachate loading had similar ammonium removal rates and nitrite accumulation but also showed higher effluent nitrate concentrations, indicating decreased denitrification levels. The SBRs adapted to 15% leachate loading had the least amount of TIN-N removal. This is likely due to the low concentrations of VSS present in the SBRs in this phase. These observations suggest that SBRs adapted to a 5% and 10% leachate loading in Phase II accomplished nitritation (first step of nitrification where ammonium is converted to nitrite) more effectively than the control in Phase III which could suggest previous exposure to leachate allowed the microorganisms to adapt to the components of the leachate that may have otherwise decreased nitritation efficacy as seen in the control. There appears to be inhibition in nitratation (second step of nitrification where nitrite is converted to nitrate), as indicated by the small accumulation of nitrite in reactors exposed to 0% leachate and 20% leachate in Phase II. There are several reasons for observed inhibition in leachate loadings over 10%, including unfavorable C:N ratios, leachate toxicity, and elevated free ammonia levels (Brennan et al, 2017). All reactors appear to have 16

30 COD (mg/l) accomplished denitrification at similar levels, indicated by the nitrate concentrations in the effluent COD Distribution - Phase III , , , , , , , , , , , % 5% 10% 15% 20% 100% Leachate Volumetric Loading Rate Adapted To (%) Effluent COD COD Removed Figure 8. COD in effluent of each SBR with the leachate loading condition that each reactor was adapted to. Bars show relative percentage of COD in the effluent, as well as any COD removal. SBRs previously exposed to leachate loadings had a higher removal of COD that the SBRs never exposed to leachate (Figure 8). The SBRs exposed to 5% and 10% leachate loading exhibited the highest amount of COD removal while the SBRs adapted to a 15% and 20% leachate loading exhibited slightly lower amounts of COD removal compared to the reactors adapted to 5% and 10% leachate loading. It can be seen that the SBRs previously exposed to 5% and 10% leachate had higher removals of TIN-N per mg of COD than the control while SBRs previously exposed to 15% and 20% did not show a significant difference of removal of TIN-N per mg of COD compared to the control (Figure 9). These findings may suggest that if BNR activated sludge is adapted to relatively low leachate loadings (5% - 10%), the sludge may be better able to handle shock loadings of leachate or temporary high loadings than if the sludge has never been exposed to leachate. 17

31 mg N Removal per mg of COD Loading mg TIN-N Removal per mg of COD Loading in Phase III % 5% 10% 15% 20% 100% Leachate Loading Adapted To (%) Figure 9. Removal of TIN-N per mg of COD loading in Phase III for each leachate loading condition. Specific Oxygen Uptake Rate (SOUR) of SBR MLSS in Phase III. Table 7 and Figure 10 show the SOUR Index for each condition. Two days into the leachate loading of 30%, the SBRs conditioned to 10% and 15% had SOUR index values higher than 1, indicating greater oxygen uptake rate per g of VSS and increased sludge activity as compared to the control. SBRs exposed to 5% and 20% in Phase II had relatively low SOUR index values two days into Phase III, indicating decreased sludge activity as compared to the control, but these SOUR index values raised after additional days of exposure. Four and seven days into the leachate loading of 30%, all SBRs had SOUR values greater than 1, indicating greater oxygen uptake rate per g of VSS and increased sludge activity as compared to SBRs never exposed to leachate. These findings suggest that previous exposure to leachate allowed the microbial community to continue functioning (indicated by oxygen uptake rate) at a higher rate than the control which had previously not be exposed to leachate. This may suggest that prior exposure to leachate will allow a microbial community to continue to survive and function at better rates when exposed to high concentrations of leachate as compared to microbial communities that have never been exposed to leachate. Figure 10 shows that, generally, each SBR previously exposed to leachate had increasing SOUR values over the 7 days, indicating increasing tolerance of the microbes to the leachate loading, as compared to the control. 18