Denitrification by Zero-Valent Iron-Supported Mixed

DENITRIFICATION BY ZERO-VALENT IRON-SUPPORTED MIXED

CULTURES

Inyoung Kim

A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Civil Engineering

Fall 2018

DENITRIFICATION BY ZERO-VALENT IRON-SUPPORTED MIXED

CULTURES

Inyoung Kim

Approved: ______Sue McNeil, Ph.D. Chair of the Department of Civil and Environmental Engineering

Approved: ______Levi T. Thompson, Ph.D. Dean of the College of Engineering

Approved: ______Douglas J. Doren, Ph.D. Interim Vice Provost for Graduate and Professional Education

I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy.

Signed: ______Daniel K. Cha, Ph.D. Professor in charge of dissertation

Signed: ______Julia Anne Maresca, Ph.D. Member of dissertation committee

Signed: ______Pei C. Chiu, Ph.D. Member of dissertation committee

Signed: ______Jeffry Fuhrmann, Ph.D. Member of dissertation committee

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my advisor, Dr. Daniel K. Cha for his continuous guidance, assistant, and expertise that I needed during my degree. I gratefully acknowledge my Ph.D. committee members, Dr. Julia A. Maresca, Dr. Pei C. Chiu, and Dr. Jeffry Fuhrmann for their time and insightful feedback for my research and thesis. My sincere thanks also goes to Mr. Michael Davidson for his tremendous technical and substantial support for research and safety in the laboratory. I am thankful to Mrs. Christine Reoli, a graduate academic advisor of the department, for her guidance and support for better graduate school life. I also thank lab manager, Dr. Yu-han Yu, and staffs of the Department of Civil and Environmental Engineering, Mrs. Karen Greco, Sarah Palmer, and Christine Murray, for their tremendous help and support. The members of the Cha group have contributed immensely to my personal and professional time at UD. The members have been good friends as well as collaborators. All past and present group members that I have had the pleasure to work with are undergraduate and graduate students Beom-seok Kim, Taylor Smith, Philip McGuire, Yaseen Al-Qaraghuli, Xiangmin Liang, Larissa Gaul, Mingjun Shao, Aidan Meese, and Rachel Aukamp; and the numerous visiting scholars who have come through the lab. In regard to the technical support, I thank the staffs Deborah Powell at UD Bioimaging Center and Drs. Choaying Ni and Yong Zhao at Keck Center for

iv Advanced Microscopy and Microanalysis for advising to acquire confocal and scanning electron microscopic images. Also, I thank Brewster F. Kingham at UD Sequencing & Genotyping Center and Dr. John Hanson, Rocio Navarro, and Lars Koenig at RTL genomics to help me to better understand microbial sequencing area. I am deeply thankful to my family: my parents and to my brother for supporting me spiritually throughout my life in general. Most of all for my loving, supportive, and encouraging husband Minho whose faithful support during the Ph.D. is appreciated.

v TABLE OF CONTENTS

LIST OF TABLES ...... ix LIST OF FIGURES ...... x ABSTRACT...... xii

Chapter

1 INTRODUCTION ...... 1

1.1 Nitrate ...... 1 1.2 Nitrate Treatment Techniques ...... 2 1.3 Objectives ...... 3

2 LITERATURE REVIEW ...... 6

2.1 Hydrogenophillic Denitrifiers...... 9 2.2 Ferrous-oxidizing Denitrifiers ...... 10 2.3 Anaerobic Ammonium Oxidation (Anammox) ...... 11 2.4 Microbial-meditated Corrosion and Surface Colonization ...... 12

3 MICROBIAL COMMUNITY ANALYSIS OF DENITRIFYING CULTURES GROWN ON ZERO-VALENT IRON ...... 14

3.1 Introduction ...... 15 3.2 Materials and Methods ...... 19

3.2.1 Microorganisms and Chemicals ...... 19 3.2.2 Denitrification Test with Various Electron Donors ...... 19 3.2.3 Chemical Analysis ...... 20 3.2.4 DNA Extraction and Sequencing for Microbial Identification ... 21 3.2.5 Bacterial 16S rRNA Gene Sequence Analysis ...... 21 3.2.6 Statistical Analysis with Sequence Results ...... 22

3.3 Results and Discussion ...... 22

3.3.1 Nitrate Reduction in Batch Reactors ...... 22 3.3.2 Microbial Diversity under Different Conditions ...... 24 3.3.3 Principal Component Analysis ...... 27

vi 4 EFFECT OF LOW TEMPERATURE ON ABIOTIC AND BIOTIC NITRATE REDUCTION BY ZERO-VALENT IRON ...... 38

4.1 Introduction ...... 39 4.2 Materials and Methods ...... 42

4.2.1 Chemicals and Microorganisms ...... 42 4.2.2 Batch Reduction Experiments ...... 43 4.2.3 Analytical Procedures ...... 43

4.3 Results and Discussion ...... 44

4.3.1 Effects of Temperature on Nitrate Reduction by ZVI ...... 44 4.3.2 Effects of Temperature on Nitrate Reduction by ZVI and Microorganisms ...... 45 4.3.3 Enhanced Denitrification by Microbial Induced Corrosion ...... 46

4.4 Conclusion ...... 48

5 NITROGEN REMOVAL BY MICROBIALLY-COLONIZED IRON GRANULES: ANAMMOX-LIKE PROCESS ...... 57

5.1 Introduction ...... 58 5.2 Materials and Methods ...... 61

5.2.1 Batch Reduction Tests with Anammox Mixed Cultures ...... 61 5.2.2 Denitrification in Anaerobic Fluidized Bioreactor ...... 63 5.2.3 Surface Study of Zero-valent Iron ...... 64

5.2.3.1 Confocal Microscopy ...... 64 5.2.3.2 Scanning Electron Microscopy ...... 65

5.2.4 Identification of Bacterial Community Colonizing Zero-valent Iron ...... 65

5.3 Results and Discussion ...... 66

5.3.1 Anammox Activities in the Presence of Zero-valent Iron: Preliminary Tests ...... 66 5.3.2 Enhanced Nitrate Reduction in Anaerobic Fluidized Bioreactor 67

5.3.2.1 Characterization of Biofilms on Zero-valent Iron ...... 68 5.3.2.2 Diversity of Microbial Communities in the Biofilm .... 69

6 CONCLUSIONS ...... 85

vii 6.1 Summary of Results ...... 85

6.1.1 Denitrification Performances in the Mixed-culture System with Various Electron Donors and Zero-valent Iron ...... 85 6.1.2 Enhanced Nitrate Removal by Microorganisms and Zero- valent Iron under Low Temperature ...... 86 6.1.3 Enhanced Nitrate Reduction by Anammox Bacteria in Iron- supported System ...... 86 6.1.4 Microbial colonization on the Surface of Zero-valent Iron ...... 86

6.2 Recommendations for Future Work ...... 87

REFERENCES ...... 89

viii LIST OF TABLES

Table 3.1. The electron donors and carbon sources of the batch experiments ...... 28

-1 Table 4.1. Estimated the effects of temperature on kobs (day ) for abiotic nitrate reduction by ZVI ...... 50

ix LIST OF FIGURES

Figure 1.1. A schematic illustration of potential pathways of nitrate reduction in the microbial-mediated zero-valent iron (ZVI) system...... 5

Figure 3.1. Nitrate removals in batch reactors under the different conditions...... 29

Figure 3.2. Nitrate removals in H2-fed microbial culture...... 30

Figure 3.3. Nitrate removals in Fe2+-fed microbial culture...... 31

Figure 3.4. Nitrate removal in ZVI-mediated microbial culture...... 32

Figure 3.5. Krona graphs of microorganisms content in seed culture identified by 16S rRNA gene sequencing...... 33

Figure 3.6. Krona graphs of microorganisms content in ZVI-supported reactor culture identified by 16S rRNA gene sequencing...... 34

Figure 3.7. Krona graphs of microorganisms content in H2-fed reactor culture identified by 16S rRNA gene sequencing...... 35

Figure 3.8. Krona graphs of microorganisms content in Fe2+-fed reactor culture identified by 16S rRNA gene sequencing...... 36

Figure 3.9. The principal component analysis (PCA) plot...... 37

Figure 4.1. Nitrogen concentrations of the unbuffered ZVI batch reactors operated o - + at 25, 17, 10, and 3.5 C: (a) NO3 , and (b) NH4 concentration...... 51

Figure 4.2. The estimation of nitrate reduction rate by ZVI under unbuffered abiotic condition at 3.5, 10, 17, and 25 oC...... 52

Figure 4.3. Arrhenius plots of nitrate removal by ZVI in batch reactors...... 53

Figure 4.4. Nitrogen concentrations of the unbuffered microbial-ZVI batch o - + reactors operated at 25, 10, and 3.5 C: (a) NO3 , and (b) NH4 concentration...... 54

x Figure 4.5. Soluble iron concentration including ferrous (Fe2+) and ferric (Fe3+) iron from batch reactors after 6 days. (a) reactor with ZVI only; (b) reactor with ZVI and cell...... 55

Figure 4.6. Schematic illustration of possible pathway of abiotic and biotic nitrate reduction by zero-valent iron (ZVI)...... 56

Figure 5.1. A schematic diagram of anaerobic fluidized bioreactor (AFBR) for denitrification test...... 73

Figure 5.2. Nitrogen profile of the anammox-ZVI batch reactor...... 74

Figure 5.3. Nitrogen concentration profile of AFBR...... 75

Figure 5.4. Confocal scanning laser microscopy (CSLM) images of ZVI surface retrieved from AFBR after 50 days operation. (a) low magnification. (b) high magnification. Bar, 5 µM...... 76

Figure 5.5. Scanning electron microscopy (SEM) image of ZVI surface retrieved from AFBR after 50 days operation...... 77

Figure 5.6. Scanning electron microscopy (SEM) image of fresh ZVI surface...... 78

Figure 5.7. The concentration of extracted genomic DNA from centrifuged pellet and ZVI surface...... 79

Figure 5.8. Krona chart of the sequencing results of bacterial taxonomic levels of extracted gDNA from the seed culture...... 80

Figure 5.9. Krona chart of the sequencing results of bacterial taxonomic levels of extracted gDNA from AFBR (after 50 days operation)...... 81

Figure 5.10. Relative phyla abundance of sequenced gDNA samples from seed culture and AFBR...... 82

Figure 5.11. Relative order abundance in phyla Proteobacteria of sequenced gDNA samples from seed culture and AFBR...... 83

Figure 5.12. Schematic illustration of the surface of ZVI and microbial colonization for enhanced denitrification with anammox...... 84

xi ABSTRACT

Microbial denitrification is an environmentally beneficial process that removes nitrate to innocuous nitrogen gas (N2) from water environment. In this study, we examined the feasibility of zero-valent iron (ZVI) as the sustained source of electron donors to support autotrophic denitrification. It was hypothesized that ZVI granules can serve as support media for the enrichment of autotrophic denitrifying population by (1) continuously supplying the electron donors via anaerobic corrosion of ZVI

2+ (e.g., cathodic hydrogen (H2) gas and ferrous (Fe ) ion), and (2) providing a nutrient- and substrate-rich solid-liquid interface for bacterial colonization. The objectives of this study were 1) to investigate the synergetic effect of denitrifying mixed cultures on nitrate reduction by ZVI, 2) to evaluate the nitrate reduction by ZVI system at low temperatures, and 3) to investigate the potential occurrence of anammox-like process in microbial-ZVI systems. A series of batch denitrification tests were conducted with ZVI granules or its

2+ corrosion products (H2 and Fe ) as the source of electron donor to elucidate the effects of these secondary electron donors on the rate and extent of nitrate reduction in the ZVI-supported mixed cultures. The ZVI-supported mixed cultures completely removed 40 mg/L of nitrate in batch reactors in 24 hours. Slower removal rates were

+2 observed in H2-cell and Fe -cell reactors as the complete removal occurred in 3 and 4 days, respectively. Repeated spiking of nitrate to batch reactors containing ZVI granules and microorganisms showed that complete nitrate reduction by the ZVI- supported cultures was sustained over a long period. In order to understand the major

xii microbial reaction in the ZVI- supported cultures, bacterial 16S ribosomal RNA (16S rRNA) gene sequencing and analysis were performed for denitrifying cultures. Analysis of microbial distribution patterns and subsequent principal component analysis (PCA) showed clear distinctions not only between ZVI-supported denitrifying culture and seed bacteria, but also among denitrifying cultures receiving different electron donors. On the other hand, the microbial composition of H2-fed cultures was similar to that of ZVI-supported cultures, suggesting that even though the hydrogen sources are different between two cultures, the same populations of bacteria may be involved in denitrification process. The effects of temperatures on abiotic and biotic nitrate reduction by zero- valent iron were examined at temperatures below 25 ℃. Under anoxic conditions,

- NO3 reduction rates in both ZVI-only and ZVI-cell reactors declined as temperature decreased. In ZVI-only reactor, 61.3% and 17.3% of initial nitrate concentration were reduced in 6 days at 25 and 3.5 ℃, respectively. The reduced nitrate was completely

+ recovered as ammonium ions (NH4 ) at both temperatures. Nitrate in the ZVI-cell reactors was completely removed within 1 – 2 days at 25 and 10 ℃, and 67 % of reduction was achieved at 3.5 ℃. Less than 25% of the reduced nitrate was recovered

+ 2+ 3+ as NH4 in all ZVI-cell reactor. Soluble iron concentrations (Fe and Fe ) in the ZVI reactors were also measured as the indicators of anaerobic corrosion. In ZVI-cell reactors, the detected soluble iron concentrations were 1.7 times higher than that in ZVI-only reactors at 25 ℃, suggesting that the enhanced nitrate reduction in the ZVI- cell reactors may be partly due to increased redox activity (i.e., corrosion) on iron surfaces. Anaerobic corrosion of ZVI was also temperature-dependent as substantially lower concentrations of corrosion products were detected at lower incubation

xiii temperatures; however, microbially induced corrosion (MIC) of ZVI was much less impacted at lower temperatures than abiotic ZVI corrosion. This study demonstrated that ZVI-supported microbial process (i.e., denitrification) is not only more sustainable at lower temperatures, but it becomes more dominant reaction for nitrate removal in microbial-ZVI systems at low temperatures. Electron donor (ammonia) and acceptor (nitrite) for the anammox reaction are typically present in microbial-ZVI systems. In this study, it was hypothesized that ZVI granules could serve as support media for the enrichment of anammox biofilm. The feasibility of ZVI-supported anammox process was demonstrated in a preliminary batch study. An anaerobic fluidized bioreactor (AFBR), using ZVI granules as the solid support materials for the biomass was operated for 50 days to study the potential colonization and stable biofilm formation of ZVI surfaces by anammox-performing cultures. During the 50-day experimental period, nitrate was completely removed with minimal production of ammonium ions. The confocal images showed that microbial colonies were formed not only at the ZVI surface, but also within the crevices of ZVI particles. Scanning electron micrograph of the ZVI granule from the AFBR shows that the ZVI surface was completely covered by the deposition of microorganisms and minerals. Genomic analysis of biofilm on the ZVI surface was conducted to examine the diversity and abundance of microbial communities in the ZVI-supported biofilm. Even though the relative abundance of Planctomycetes in the ZVI culture was small, the detection of Planctomycetes combined with the chemical data supporting anammox-like process suggested that low ammonia accumulation in the AFBR may be due to anammox activity.

xiv

Chapter 1

INTRODUCTION

This chapter will introduce the concern of nitrate contamination of the aquatic environment and the application of zero-valent iron (ZVI)-supported denitrification as an effective treatment technology for nitrate contamination. The specific objectives of this research will be also outlined in this chapter.

1.1 Nitrate

- Nitrate (NO3 ) is a common contaminant of ground and surface water from anthropogenic activities, such as agricultural runoff, and industrial and domestic effluent discharge (Spalding and Exner, 1993; Holloway et al., 1998; Ward et al., 2005; Ghafari et al., 2008). Increased discharge and the accumulation of nitrate in water bodies cause eutrophication (McIsaac et al., 2001). Moreover, the excessive

- - amounts of NO3 in drinking water can be potentially transformed to nitrite (NO2 ) in humans, and result in methemoglobinemia especially among infants. Methemoglobinemia is a condition in which hemoglobin is oxidized to methemoglobin by nitrate or nitrite and loses its ability to bind and transport oxygen (Mueller and Helsel, 1996; Wright et al., 1999; Shirimali and Singh, 2001). Nitrite is also linked to the formation of carcinogenic nitrosamines from reaction with secondary or tertiary amines (Doyle et al., 1997; Glass and Silverstein, 1999). For these reasons, US Environmental Protection Agency (US EPA) sets the maximum

- contaminant level (MCL) of nitrate in drinking water as 10 mg NO3 -N/L to avoid the potential health problems (Kapoor and Viraraghavan, 1997).

1.2 Nitrate Treatment Techniques The conventional approaches to eliminate nitrate from water include ion exchange, reverse osmosis, and electro-dialysis. These processes have been limited as they are relatively expensive and these processes only separate nitrate from one liquid phase to concentrate them in another (Shrimali and Singh, 2001). In addition, subsequent treatment of concentrated waste brine results in an additional disposal problems (Ergas and Rheinheimer, 2004). Biological denitrification is an alternative approach for nitrate removal, which transforms nitrate to innocuous nitrogen gas (N2) under anaerobic conditions. This process is an attractive treatment option due to the high specificity of denitrifying bacteria for nitrate, lower cost, and higher removal rates than other processes (Matéju et al., 1992). Denitrification is the process involves the reduction of the oxidized forms of nitrogen coupled with the oxidation of electron donors (Payne, 1976). As electron

2+ donors, organic compounds, hydrogen gas (H2), ferrous iron (Fe ), and sulfur have been extensively studied (Nielsen and Nielsen, 1998; Zhang and Lampe, 1999; Haugen et al., 2002; Heylen et al., 2006). An alternative application that may serve as a source of multiple electron donors for autotrophic denitrification process is zero-valent iron (ZVI). Anaerobic corrosion of ZVI produces hydrogen gas through the reduction of protons as well as ferrous irons. Both corrosion products can be utilized by denitrifying bacteria, and both of reactions are thermodynamically favorable (Figure 1.1) (Straub et al., 1996; Liu et al., 2006; Shin and Cha 2008; Zhang et al., 2014). In addition, ZVI application

to hydrogen-utilizing denitrifying system is safer and less expensive way than directly applying dangerous hydrogen gas (Choe et al., 2000). On the other hand, the reductive treatment of nitrate in the presence of ZVI

+ generates ammonium ion (NH4 ) as by-product which may limit widespread environmental applications. One way to overcome this accumulation of ammonia in the nitrate-reducing ZVI system is to introduce anammox process. Anaerobic ammonium oxidation (anammox) process is the conversion of ammonium and nitrite into nitrogen gas (van de Graaf et al., 1996). This is an important microbial pathway in the nitrogen cycle because it shortens the pathways of typical ammonium removal (nitrification) and nitrite reduction (denitrification) processes. It also does not require any external electron donors (Ni et al., 2010). ZVI system may provide a favorable environment for anammox bacteria due to the presence of: (1) ammonia as a product of abiotic nitrate reduction and (2) nitrite as an intermediate of denitrification.

1.3 Objectives The goal of this study was to evaluate the feasibility of zero-valent iron to support microbial denitrification for the treatment of nitrate contamination. The specific objectives were: 1) To investigate the synergetic effect of denitrifying mixed cultures on nitrate reduction by ZVI. Batch denitrification tests were conducted with

2+ ZVI granules or its corrosion products (H2 and Fe ) as the source of electron donor to elucidate the role of different ZVI-associate electron donors on the rate and extent of nitrate reduction in the ZVI-supported mixed cultures. In addition, genomic analysis coupled with chemotaxonomic assays was conducted to identify microbial compositions

in denitrifying mixed cultures utilizing different electron donors. Dominant microbial populations were also identified to elucidate the primary pathway for nitrate removal. 2) To evaluate the nitrate reduction by ZVI system at low temperatures. Emphasis was on investigating the role of microorganisms and effect of temperature on nitrate removal efficiency and ammonia production. The study also examined microbially induced corrosion process at the ZVI surfaces to elucidate the interactions and synergies between ZVI and microorganisms in microbial-ZVI systems at temperatures below 25 ℃. 3) To investigate the potential occurrence of anammox-like process in microbial-ZVI systems. An anaerobic fluidized bioreactor (AFBR), using ZVI granules as the solid support materials for the biomass was operated to study the potential colonization and stable biofilm formation of ZVI surfaces by anammox-performing cultures. The confocal and scanning electron microscopy were used to characterize microbial colonies and biofilm formation on the ZVI surface. Genomic analysis of biomass in AFBR was conducted to examine the diversity and abundance of microbial communities in the ZVI-supported biofilm.

Anoxic

H - - 2 NO3 NO3

0 Fe 2+ Abiotic Fe Biotic

H2O

+ - NH4 NO2

N N 2 2 Denitrifying & Anammox Bacteria

Figure 1.1. A schematic illustration of potential pathways of nitrate reduction in the microbial-mediated zero-valent iron (ZVI) system.

Chapter 2

LITERATURE REVIEW

- This chapter provides an overview of microbial-mediated nitrate (NO3 ) reduction processes supported by zero-valent iron (ZVI) in water. The first part of literature includes a review of conventional physico-chemical technologies to eliminate nitrate and their application limits. Then, a body of literature on the potential autotrophic denitrification that can be enhanced by the presence of ZVI is summarized in detail. Additional supportive concepts and theories that help to understand the mechanisms in the ZVI-amended mixed culture system are also covered. Nitrate ions are not readily removed by common water treatment technology, such as coagulation, oxidation, and filtration, because of the stability and high solubility of nitrate with low potential for precipitation or adsorption. Nitrate- contaminated water is typically treated by ion exchange, reverse osmosis, electrodialysis, and chemical or biological denitrification (Kapoor and Viraraghavan, 1997; Shrimali and Singh, 2001). Ion exchange process uses anion resins for nitrate removal. The nitrate- contaminated water pass through a resin bed, and nitrate ions are exchanged for chloride until the capacity of resin is exhausted (Clifford and Liu 1993; Kim and Benjamin, 2004). This process generally achieves nitrate levels lower than the maximum contaminant level (MCL) of US EPA (US EPA, 1982). However, the presence of other anions, especially sulfate, can deteriorate the nitrate removal efficiency of ion exchange process (Cheng et al., 1997; Kapoor and Viraraghavan,

1997). To enhance efficiency of nitrate elimination, a nitrate-preferred resin was developed, but this resin still requires frequent regeneration and high maintenance cost (Viraraghavan, and Corkal, 2003; Ergas and Rheinheimer, 2004). Reverse Osmosis (RO) is another common method for removing nitrate from water supplies. Common problems of reverse osmosis process are membrane fouling and deterioration with time due to the deposition of soluble materials, organic matters, and particles (Kapoor and Viraraghavan, 1997). In addition, RO process potentially generates large waste volumes requiring proper disposal (low water recovery) and requires high operation and maintenance costs (Elmidaoui, et al., 2001; Jensen et al., 2012). In addition to ion exchange and reverse osmosis, electro-dialysis is another physical-chemical process that has been used to treat nitrate-contaminated water by selective removal of undesirable nitrate by the passage of a direct electric current (Miquel and Oldani, 1991; Hell et al., 1998). Electro-dialysis requires higher maintenance cost for efficient operation. Compared to reverse osmosis, it requires more pretreatment to avoid fouling, and waste disposal (Kapoor and Viraraghavan, 1997; Wąsik et al., 2001; Jensen et al, 2012). Biological denitrification is microbially-mediated nitrate reduction, which ultimately produces innocuous nitrogen gas (N2) under anaerobic conditions. Denitrification proceeds in a series of intermediate reactions producing gaseous nitrogen (Payne, 1976; Knowles, 1982):

- - NO3 → NO2 → NO → N2O → N2 (2.1) A variety of different electron donors have been shown to support the growth of denitrifying bacteria (Nielsen and Nielsen, 1998; Zhang and Lampe, 1999; Haugen

et al., 2002; Elefsiniotis et al., 2004; Hallin et al., 2006; Cyplik et al., 2012). These include ethanol, fatty acids, and vegetable oils. Acetate has been commonly used as an exogenous electron donor for heterotrophic denitrification in environmental applications (Heylen et al., 2006). In comparison to heterotrophic denitrification, autotrophic denitrification processes have two advantages: (1) no need for an external organic carbon sources (e.g., methanol, acetate, etc.), thereby lowering the cost, (2) less sludge production which minimize the handling of excessive sludge (Koenig and Liu, 1996; Till et al., 1998; Zhang and Lampe 1999; Oh et al., 2003). Autotrophic denitrifiers require inorganic carbon as carbon source and utilize electron donors, such as reduced sulfur

2+ compounds, H2, uranium (U(IV)), ferrous iron (Fe ) (Nielsen and Nielsen, 1998; Zhang and Lampe, 1999; Haugen et al., 2002; Beller, 2005; van Rijn et al., 2006). Zero-valent iron has been applied to various environmental treatment processes as a strong reducing agent (E0 = -0.44 V). Tetrachloroethylene (TCE) can be easily dechlorinated by ZVI in both batch-scale and field-scale tests (Chen et al., 2001; Ibrahem et al., 2012). A chromate-contaminated groundwater can be remediated by ZVI packed permeable reactive barrier. Through this barrier, Cr(VI), which is extremely toxic and carcinogenic (De Flora et al., 1990), is converted into Cr(III), which is actually essential nutrient for human (Mertz, 1981). ZVI also reduces ammunition, such as 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5- triazine (RDX) from groundwater (Oh et al., 2003). The nitro group of these chemicals are reduced into amino group, which are more susceptible for subsequent treatments. ZVI also has been used successfully for abiotic nitrate reduction from water in lab-scale tests (Siantar et al., 1996; Cheng et al., 1997; Choe et al., 2004). Abiotic

reduction of nitrate by ZVI is thermodynamically favorable under anaerobic conditions (Westerhoff, 2003, Choe et al., 2004). In addition, ZVI systems require less maintenance cost (Muegge, 2009), and has relatively innocuous byproduct from the reaction (Gavaskar et al., 2005) compared to ion exchange or reverse osmosis. However, reductive treatment of nitrate with ZVI possesses many drawbacks requiring acidic conditions. This process also requires further treatment of ferrous ion and ammonia which is a main product of reduction (Huang et al., 1998; Alowitz and Scherer, 2002). Alternatively, several studies have shown that ZVI can support microbial reduction of nitrate to nitrogen gas (Till et al., 1998; Choe et al., 2000; Gu et al., 2002; Biswas and Bose, 2005; Shin and Cha, 2008). Till and colleagues (1998) showed that zero-valent iron can serve as electron donor for nitrate reduction by providing hydrogen to the nitrate respiring bacteria. They suggested that the use of ZVI could eliminate the need to continually supply expensive electron donors or explosive

- hydrogen gas. Schaefer et al. (2007) reported complete removal of 4 mg/L of NO3 -N with ZVI and microbial culture. Potentially, there are multiple possible microbial communities that may be responsible for nitrate reduction in ZVI system. These includes (1) hydrogenophillic denitrifiers, (2) ferrous-oxidizing denitrifiers, and (3) anammox bacteria.

2.1 Hydrogenophillic Denitrifiers Hydrogenophillic denitrifiers utilize cathodic hydrogen gas generated from the anaerobic corrosion of ZVI in water through batch tests (Shin and Cha, 2008):

0 2+ - Fe + 2 H2O  Fe + H2 + 2 OH (2.2)

- - 2 NO3 + 5 H2  N2 + 4 H2O + 2 OH (2.3)

Hydrogen is one of the most thermodynamically favorable electron donors for nitrate reduction (Till et al., 1998). H2 is excellent electron source because of its clean nature, low biomass yield which requires further steps of treatment. Moreover, the reaction products of hydrogenophilic denitrification are innocuous (N2 and water) (Smith et al., 1994; Vasiliadou et al., 2006; An et al., 2010). Nevertheless, the application of hydrogen gas is limited due to relatively high cost, and its explosive properties (Kielemoes et al., 2000; Biswas and Rose, 2005).

ZVI has been suggested as a safe and continuous source of H2 for hydrogenophilic denitrification activity. It was confirmed that ZVI produces cathodic hydrogen under anaerobic conditions (Weathers et al., 1997). Till et al. (1998) demonstrated the feasibility of supporting autotrophic denitrification using steel wool. While all of nitrate in steel wool only system was recovered as ammonia, microbial- ZVI system showed only 28% of ammonia.

2.2 Ferrous-oxidizing Denitrifiers Ferrous-oxidizing denitrifiers use ferrous ions as electron donor to reduce nitrate (Straub et al., 1996). Ferrous ions are abundantly produced from ZVI corrosion under anaerobic conditions (Equation 2.2, Enning and Garrelfs, 2014). There was geochemical evidence that nitrate serves as an oxidant for ferrous iron in anoxic environment, based on the chemical gradients (Froelich et al., 1979). And then, there has been several description for nitrate-reducing, iron-oxidizing bacteria. Straub et al. (1996) studied the microbial oxidation of ferrous iron coupled with nitrate reduction under anoxic conditions. They obtained the first ferrous- oxidizing, nitrate-denitrifying culture from brackish water lagoon sediment. This

culture grew autotrophically, using ferrous iron as the sole electron donor. The result showed good agreement with the expected stoichiometry of the following equation:

2+ - 3+ + 5Fe + NO3 + 12H2O → 5Fe + 0.5 N2 + 9H (2.4) Nielsen and Nielsen (1998) demonstrated the occurrence of microbial ferrous iron-dependent nitrate removal in activated sludge simultaneously with the oxidation of Fe2+ to Fe3+ iron. It showed agreement of stoichiometric equation with what Straub et al. (1996) explained. Zhang et al. (2014) isolated a denitrifying bacterium which utilize ferrous iron as electron donor from deep sediment of lake. The main product of Fe2+-dependent nitrate removal was dinitrogen, as there was not any accumulation of ammonia, nitrous oxide, or nitrite.

2.3 Anaerobic Ammonium Oxidation (Anammox) Anammox bacteria utilize ammonia as electron donor and nitrite as final electron acceptor (van de Graaf et al., 1996).

+ - NH4 + NO2 → N2 + 2H2O (2.5) Anammox reaction was firstly observed in an autotrophic denitrification reactor with the presence of ammonium under sulfide-limiting condition (Mulder et al., 1995). Anammox process is performed by bacteria belonging to phylum Planctomycetes (Strous et al., 1999) containing membrane-bound organelle which is responsible for ammonium and nitrite conversion to nitrogen gas (Damsté et al., 2002). Bacteria that are responsible for anammox reaction are characterized by extremely slow growth rate (Strous et al., 1998). Anammox is promising process in terms of energy saving and efficient nitrogen removal (Kuenen, 2008). Compared to traditional nitrification-denitrification process, the autotrophic anammox process consumes 100% less organic carbon and

almost 50% less energy for oxygen used for aerobic nitrification. Thus, anammox application has less sludge production and cost for operation (Tal et al., 2005; van der Star et al., 2007; Molinuevo et al., 2009). In microbial ZVI systems, ammonia is produced due to abiotic reduction of nitrate by ZVI (Cheng et al., 1997) and nitrite presents as an intermediate of hydrogenophilic autotrophic denitrification (Park et al., 2013). Thus, the coexistence of anammox process and microbial-ZVI denitrification bacteria could enhance nitrogen removal efficiency.

2.4 Microbial-meditated Corrosion and Surface Colonization Microbially induced corrosion (MIC) is a common process on the metal surface illustrating the activity of microorganisms and anaerobic corrosion of iron in the environment (Stott, 1993; Xu et al., 2013; Enning and Garrelfs, 2014). Biocorrosion accompanies by the biofilm formation, which is initiated from the cell- surface interactions in response to the metabolic redox reaction, nutrient scavenge, etc. (O’Toole et al., 2000; Fang et al., 2002; Beech and Sunner, 2004; Koch, 2014). Once bacteria associated with the metal surface, biofilm consortia would become more tight and complex with microorganisms, corrosion products, and extracellular polymeric substances (EPS) secreted by bacteria (Lee and Newman, 2003; Tuson and Wiedel, 2013). The activity of microorganisms associated with the ZVI, such as a redox reaction for bacterial growth, stimulate biocorrosion and the formation of biofilm (Beech and Sunner, 2004). Especially, there have been many studies on the microbial colonization on the ZVI surfaces by sulfate-reducing bacteria (SRB) which utilize

2- cathodic H2 gas as electron donor to reduce sulfate (SO4 ) to hydrogen sulfide (H2S)

under the anoxic condition (Videla, 2000; Beech and Campbell, 2008; Celis et al., 2009). This type of biocorrosion has been studied broadly, especially for the bioreactors where microorganisms reducing either sulfate or nitrate (Hamilton, 1985; Dowling et al., 1992; De Windt et al., 2003; Hubert et al., 2005; Mori et al., 2010; Xu et al., 2013).

Chapter 3

MICROBIAL COMMUNITY ANALYSIS OF DENITRIFYING CULTURES GROWN ON ZERO-VALENT IRON

Abstract Anaerobic corrosion of zero-valent iron (ZVI) can provide electron donors for the autotrophic microbial denitrification as cathodic hydrogen (H2) and ferrous iron (Fe2+) are the major corrosion products. In this study, we examined the feasibility of ZVI as the sustained source of electron donors for efficient and continuous treatment of nitrate contaminated water. A series of batch denitrification tests were conducted

2+ with ZVI granules or its corrosion products (H2 and Fe ) as the source of electron donor to elucidate the role of different ZVI-associate electron donors on the rate and extent of nitrate reduction in the ZVI-supported mixed cultures. ZVI-supported mixed cultures completely removed 40 mg/L of nitrate in batch reactors in 24 hours. Slower

+2 removal rates were observed in H2-cell and Fe -cell reactors as the complete removal occurred in 3 and 4 days, respectively. Repeated spiking of nitrate to batch reactors containing ZVI granules and microorganisms showed that complete nitrate reduction by the ZVI-supported cultures was sustained over a long period. In order to understand the major microbial reaction in the ZVI- supported cultures, bacterial 16S ribosomal RNA (16S rRNA) gene sequencing and analysis were performed for denitrifying cultures. Analysis of microbial distribution patterns and subsequent principal component analysis (PCA) showed clear distinctions not only between ZVI-supported denitrifying culture and seed bacteria, but also among denitrifying cultures receiving

different electron donors. On the other hand, the microbial composition of H2-fed cultures was similar to that of ZVI-supported cultures, suggesting that even though the hydrogen sources are different between two cultures, the same populations of bacteria may be involved in denitrification.

3.1 Introduction

- Nitrate (NO3 ) is one of the essential nutrients for plant and animal growth. However, the excess levels of nitrate introduction into the water environment by anthropogenic, agricultural, and industrial activities can harm water bodies and people in many ways, such as eutrophication and blue baby syndromes (Cheng et al., 1997; Fennesy and Cronk, 1997; Knobeloch et al., 2000). Therefore, the importance of efficient and effective nitrate removal from the water environment has emphasized significantly (Payne, 1976; Barber and Stuckey, 2000). Denitrification is a microbial process that reduce nitrate into innocuous nitrogen gas by utilizing nitrate as terminal electrons acceptor under the absence of oxygen (Payne, 1976; Firestone et al., 1979). Denitrifying bacteria ubiquitously exist in a wide range of environments, such as soil, marine, and freshwater sediment (Gamble et al., 1977; Inglett, et al., 2005), and they are commonly used to remove nitrogen from municipal and industrial wastewater (Chen and Lin, 1993; Rossi et al., 2015; Oh et al., 2002). Applying denitrifying bacteria for nitrate reduction is beneficial because it is biological process which is environment-friendly, and produce non-toxic compounds. Moreover, it has reasonable operating costs compared to other physical or chemical treatment of nitrate (Mateju et al., 1992; Barber and Stuckey, 2000; Kruglova et al., 2017). The following is subsequent nitrate reduction stages (Knowles, 1982);

- - NO3 → NO2 → NO → N2O → N2 (3.1) Among the various denitrifying process by bacteria, an autotrophic denitrification is preferred process due to its advantages: (1) no need to supply an external organic carbon compounds such as carbohydrates, organic alcohols, or acids (van Rijn et al., 2006) as an electron donor; and (2) less sludge production which minimizes the further treatments of sludge (Agrawal and Tratnyek, 1996; Till et al., 1998; Oh et al., 2003; Schaefer et al., 2007; Shin and Cha, 2008). For Several potential electron donors have been reported for autotrophic denitrifying bacteria previously,

2+ 2+ such as hydrogen gas (H2), ferrous iron (Fe ), manganese (Mn ), and sulfide and sulfur (Driscoll and Bisogni, 1978; Straub et al., 1996; Till et al., 1998; Sliekers et al, 2002; van Rijn et al., 2006). Zero-valent iron (ZVI) has been applied into the contaminant remediation of the environment in many ways; for example, dehalogenation of chlorinated aliphatic compounds and removal of toxic metal contaminants and organic dyes from the groundwater and surface water (Gillham and O’Hannesin, 1994; Kanel et al., 2005; Nam and Tratnyek, 2000; Ma and Zhang, 2008; Comba et al., 2011; Fu et al., 2014). It is advantageous to apply ZVI for environmental treatment because of its relative low cost as well as wide applicability with strong reducibility (Fe0 → Fe2+ + 2e-, E0 = 0.44 V, Gillham and O’Hannesin, 1994; Ponder et al., 2000; Ahn et al., 2008; Ma and Zhang, 2008). ZVI have also been used for chemical reduction of nitrate (Siantar et al., 1996; Cheng et al., 1997; Huang et al., 1998; Choe et al., 2004) which is thermodynamically favorable in the absence of oxygen (Westhoff, 2003). However, the abiotic nitrate

reduction with ZVI produces ammonia and ferrous iron which requires further treatment (Huang et al., 1998; Alowitz and Scherer, 2002):

- 0 + + 2+ NO3 + 4Fe + 10H3O → NH4 + 4Fe + 13H2O (3.2)

2+ Cathodic hydrogen (H2) and ferrous iron (Fe ) are the major products of the anaerobic corrosion of ZVI (Daniels et al., 1987; Häring and Conrad, 1991; Smith et al., 1994; Till et al., 1998):

0 2+ - Fe + 2H2O → H2 + Fe + 2OH (3.3)

Thus, it can be hypothesized that ZVI can serve as a precursor of the electron donors for the autotrophic microbial denitrification. It has been previously shown that the products of ZVI corrosion can support microbial reduction of nitrate to nitrogen gas under the anaerobic condition (Straub et al., 1996; Till et al., 1998). Therefore, there can be two potential processes by denitrifying bacteria which are utilized two different electron donors for their anaerobic respiration in ZVI-implemented system. The first potential nitrate reduction in the microbial-mediated ZVI system is hydrogenophilic denitrification, which responsible bacteria utilize hydrogen gas as electron donor for nitrate reduction:

- - 2NO3 + 5 H2 → N2 + 4H2O + 2OH (3.4) Hydrogen in one of the most thermodynamically favorable electron donors for nitrate reduction. It was confirmed that cathodic hydrogen gas (H2) from anaerobic ZVI corrosion can be utilized by pure or mixed culture of denitrifying bacteria (Smith et al., 1994; Till et al., 1998; Vasiliadou et al., 2006 Shin and Cha, 2008; An et al., 2010). Moreover, the application of ZVI as a source for hydrogen gas is beneficial because it is much cheaper and safer than using hydrogen gas (Kielemoes et al., 2000; Biswas and Bose, 2005).

Another ZVI applicable process is denitrification by ferrous-oxidizing bacteria (Straub et al., 1996):

- 2+ 3+ + NO3 + 5 Fe + 12H2O → 0.5 N2 + 5Fe + 9H (3.5) Straub and colleagues (1996) confirmed that the microbial oxidation of ferrous iron could be coupled with nitrate reduction under anaerobic conditions. They also identified the first ferrous-oxidizing, nitrate-reducing strain from the sediment of brackish water lagoon, and this strain grew autotrophically using ferrous iron as the sole electron donor. Nielsen and Nielsen (1998) also demonstrated the occurrence of microbial ferrous iron-dependent nitrate removal in activated sludge with the simultaneous oxidation of ferrous to ferric iron. The stoichiometry of the reaction agreed to the previous study. Denitrification by isolated denitrifying bacterium which use ferrous iron as electron donor from deep sediment of lake was also identified (Zhang et al., 2014). ZVI application to denitrification is an attractive way to treat nitrate because it is not only less expensive than other physico-chemical nitrate removal methods, but also sustainable with relatively less harmful by-products to the environment (Schaefer et al., 2007; Oh et al., 2016). ZVI is also continuous source of hydrogen gas and ferrous iron for long-term environmental applications (Farrell et al., 2000). In addition, the autotrophic denitrification supported by ZVI is preferred due to its less biomass production.

In this chapter, the feasibility of ZVI as a source of electron donors, H2 gas and Fe2+, which can be utilized by various denitrifying bacteria among the activated sludge was evaluated. Specifically, laboratory-scale batch tests were performed in order to assess the extent of microbial and chemical nitrate reduction in the ZVI-supported

mixed cultures. Then, the presence of denitrifying bacteria and the compositions of microbial communities in each reactor fed with different electron donors were identified by 16S rRNA gene sequencing. To understand the correlation between microbial activity depending on the electron donors and community diversity, principal component analysis (PCA) was employed.

3.2 Materials and Methods

3.2.1 Microorganisms and Chemicals Denitrifying seed cultures were obtained from activated sludge aeration basin of Wilmington wastewater treatment plant (Wilmington, DE, USA). The collected activated sludge cultures were directly used as the seed culture for batch denitrification experiments without performing prior enrichment. Concentration of seed cultures were adjusted to 1000 mg/L as total suspended solids (TSS) with the culture media. The culture medium was contained 300 mg/L of NaHCO3, 300 mg/L of

KH2PO4, 100 mg/L of MgCl2‧6H2O, 25 mg/L of CaCl2‧2H2O, and 1 ml of trace element solution. The trace element solution contained 450 mg/L of FeCl2‧4H2O, 190 mg/L of CoCl2‧6H2O, 100 mg/L of MnSO4‧7H2O, 52 mg/L of ZnCl2, 36 mg/L of

Na2MoO4‧2H2O, 30 mg/L of H3BO3, 29 mg/L of CuCl2‧2H2O, and 24 mg/L of

NiCl2‧6H2O. To maintain an optimal physiological pH between 7.2 and 7.6, 0.1 M of HEPES was added (Good et al., 1966; Shin and Cha, 2008; Saleh-Lakha et al, 2009).

3.2.2 Denitrification Test with Various Electron Donors The lab-scale denitrification batch tests were performed using 250 ml amber bottles at room temperature. The concentration of nitrate in each reactors was 40 mg/L as N. The volume of medium and 1,000 mg/L TSS of activated sludge were prepared

as 100 ml. One of the bottles contained 30 g of ZVI which was purchased from Peerless Metal Powders (Detroit, MI), and sieved with 10 – 20 mesh (obtaining particles of 0.841 – 2 mm). Two others were fed with H2 gas (compressed, 99.999% purity, BOC gases), and FeCl2 based on stoichiometry from the previous studies (Till et al., 1998; Straub et al., 1996), respectively.

The prepared solutions were degassed by purging with N2 gas at least 15 minutes, and were sealed with screw thread Mininert valve (Sigma-Aldrich, St. Louis, MO) and low-permeability tape (3M, Maplewood, MN). All preparation processes for batch tests were performed in the anaerobic chamber (Cole-Parmer, Vernon Hills, IL). Table 3.1 shows the experimental setup. The test bottles were continuously shaken on a platform shaker (New Brunswick Scientific, Edison, NJ) at 150 rpm.

3.2.3 Chemical Analysis 5 mL of samples for chemical analysis were collected in duplicate everyday by using gas-tight sampling syringe with shut-off valve (Restek, Bellefonte, PA) to prevent any additional reaction with air during sampling. The samples were filtered by WhatmanTM binder-free microfiber filters (General Electric Whatman, Marlborough, MA). Nitrate and nitrite were measured by ion chromatography (IC, ICS-1000, Thermo Fisher Scientific, Hampton, NH), and ammonia was determined by spectrophotometer (DR5000, Hach Company, Loveland, CO).

3.2.4 DNA Extraction and Sequencing for Microbial Identification After establishing sustained denitrifying activities in each bottle, the reactors were sacrificed for microbiological analysis to confirm the presence of denitrifying bacteria and the diversity of communities when they were given different electron donors for denitrification. Genomic DNA was extracted and purified by using BioReagentsTM SurePrepTM Soil DNA Isolation Kit (Fisher Scientific, Hampton, NH) following manufacturer’s protocol. DNA concentration and purity were determined using a NanoDrop ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE). The extracted genomic DNA samples were stored at -20℃ before being sent to RTL Genomics (Lubbock, TX) for 16S rRNA gene sequencing.

3.2.5 Bacterial 16S rRNA Gene Sequence Analysis Prokaryotic 16S ribosomal RNA (16S rRNA) among the extracted genomic DNA (gDNA) from each reactor was targeted for sequencing to identify and compare bacteria present within samples. The 16S rRNA amplicon was sequenced by Illumina MiSeq sequencing platform at the RTL Genomics (Lubbock, TX) upon the protocols. Including the extracted gDNA from seed culture, overall 4 samples were amplified by using the primer set 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’- GGACTACHVGGGTWTCTAAT-3’). The primer set was selected to amplify variable V4 region among the 16S rRNA gene (Caporaso et al., 2011). Illumina sequence data were obtained for the paired end reads (2×250), then demultiplexed by the Illumina software. The final data after quality checking and taxonomic identification were visualized into Krona pie graphs which represents the species diversity per sample (RTL Genomics, 2016), and sent back to us. Krona is a powerful

metagenomic visualization method which composes and displays taxonomic data and their relative abundances from the samples (Ondov et al., 2011).

3.2.6 Statistical Analysis with Sequence Results Principal component analysis was employed to identify a pattern in a data set, and compare similarity of bacterial community according to the given electron donors. The data set for PCA was prepared based on Krona graphs of bacterial 16S rRNA gene sequencing result from each reactor. A data set was composed of biological taxonomic classifications and their relative abundance. A free statistical software environment, R (R v.3.4.2; https://www.r-project.org/) was used for PCA analysis. The data sets were plotted using the first two principal components (PC) produced by prcomp function and biplot in R for data processing and visualize the data.

3.3 Results and Discussion

3.3.1 Nitrate Reduction in Batch Reactors Nitrate concentrations in batch reactors containing different electron donors are shown in Figure 3.1. Denitrifying cultures in the batch reactors completely removed

2+ nitrate within 2 - 4 days when they were fed with ZVI, H2 gas or Fe ions. Nitrate (40 mg/L-N) in the reactors containing both ZVI granules and cells was completely removed to below the detection limit (below 0.3 mg/L) in 24 h. In contrast, only 32.9% of the initial nitrate was removed in the ZVI-only reactors (no cells) over 3 days, indicating iron alone was not effective in removing nitrate. Nitrate reduction in the cell control, which contained the seed culture but no external electron source (i.e.,

2+ no added ZVI, H2, or Fe ), was slow but more noticeable than the abiotic control. The 40.8% reduction in nitrate concentration over the 3 days was most likely due to

fermentable organic materials carried over from the seed culture. Nitrate removal was not observed in the medium control containing only sterile medium under N2 gas.

+2 Nitrate was completely removed in the H2-cell and Fe -cell reactors in 3 and 4 days, respectively. The initial ferrous ion concentration was in excess of the stoichiometric requirement (120%) for complete removal of nitrate. Nitrate reduction

2+ in the ZVI-cell was substantially faster than that in H2-cell and Fe -cell reactors (Figure 3.1). This result may be due to the presence of both hydrogen and ferrous iron in the ZVI-cell reactor as reducing equivalents for denitrification reactions. In addition, the abiotic reduction of nitrate by ZVI may have contributed to the faster nitrate removal. Repetitive spiking of the experimental reactors was performed to assess if denitrification reactions can be sustained over multiple feeding cycles. Nitrate stock solution was spiked into the denitrifying reactors after the disappearance of initial nitrate. Figure 3.2 and 3.3 demonstrated that complete removal of nitrate was not only sustained throughout the experimental period but the rate of nitrate reduction was

2+ enhanced in both H2-cell and Fe -cell reactors, possibly due to the increase in the density of denitrifying populations. About 30% of removed nitrate was recovered as ammonia (nitrogen basis) in the ZVI-cell reactor after 1 day, indicating abiotic reduction of nitrate. Ammonia concentrations gradually increased in the ZVI-cell reactor over five re-spiking cycles

2+ (Figure 3.4), while no ammonia accumulation was observed in the H2- or Fe - reactors.

3.3.2 Microbial Diversity under Different Conditions Microbial genomic DNA (gDNA) including 16S rRNA genes were extracted from each denitrifying reactors using BioReagentsTM SurePrepTM Soil DNA Isolation Kit (Fisher Scientific, Hampton, NH) following manufacturer's protocol. Once the purified gDNA samples met the required concentration of 20 ng/μL and quality ratio of 1.8, the samples were sent to the RTL Genomics (Lubbock, TX) for amplification and sequencing for the bacterial and taxonomic identification. Sequencing results obtained from RTL Genomics ranged from 8,978 to 30,723 sequences per sample. The final data after quality checking and taxonomic identification, Krona chart was constructed to illustrate the microbial distribution pattern of each batch reactor (Figure 3.5 – 3.8). The results illustrates that seed cultures obtained from the activated sludge basin of Wilmington Wastewater Treatment Plant (Wilmington, DE) comprised of diverse and rich bacterial communities (Figure 3.5). At the phylum level, the most abundant bacterial populations were Proteobacteria (83% of bacteria) and Bacteroidetes (8% of bacteria). Similarly, previous studies have reported that Proteobacteria as the most dominant phylum in activated sludge samples of full-scale wastewater treatment plants (Sanapareddy et al., 2009; Zhang, et al., 2012). The most abundant class was Betaproteobacteria (57%), of which Rhodocyclales and Burkholderiales were the dominant taxa at the order level. Betaproteobacteria have been often reported as the dominant activated sludge communities (Yu and Zhang, 2012). Further affiliation revealed that the most abundant genus in the seed cultures was Zoogloea (20%) of Rhodocyclaceae family. Zoogloea sp. is commonly detected floc-forming bacteria in activated sludge (Rossello-mora et al., 1995; Wagner et al., 2002).

The composition of bacterial communities enriched by ZVI as the primary electron donor is shown in Figure 3.6. The sequencing results indicate that ZVI- enriched cultures were again dominated by Proteobacteria, followed by Firmicutes. The abundance at the class and order levels did not vary considerably after multiple cycles of ZVI enrichment. The most abundant class was Betaproteobacteria, of which Rhodocyclales and Burkholderiales were the dominant taxa at the order level, which are similar to the seed cultures. At the genus level, however, a significant shift was observed. After the ZVI enrichment, the relative abundances of Dechloromonas sp. and Azospira sp. increased sharply to 27% and 9%, respectively (Figure 3.6). Dechloromonas sp. has been reported as one of the common denitrifying bacteria in full-scale wastewater treatment plants (Zienlinska et al., 2016). Azospira has been previously reported to have nitrate reduction and nitrogen-fixing capabilities (Bae et al., 2007; Oosterkamp et al., 2011).

In the H2-cell reactor, the phyla of Proteobacteria and Bacteroidetes dominated (Figure 3.7), which are similar to that of seed cultures. At the genus level,

Dechloromonas sp. dominated at the end of H2-fed cultivation with a high relative abundance of 38%. Sharp increase in the relative abundance of the well-known denitrifier, Dechloromonas sp., along with rapid nitrate reduction in the H2-fed cultures suggests that Dechloromonas sp. may play an important role in nitrate reduction reaction with hydrogen as the primary electron donor. Dechloromonas sp. belongs to the Rhodocyclaceae family under the Betaproteobacteria class. Hydrogen oxidizing betaproteobacterial denitrifiers are known to produce N2 rather than ammonium as the end product of nitrate reduction (Flynn et al., 2014), which is consistent with our results as no ammonium was detected in the H2-cell reactor.

For the Fe2+-cell reactor, a significant shift in the microbial composition was observed at the class level after the Fe2+-fed cultivation under anoxic conditions. From the seed cultures to Fe+2-fed cultures, the relative abundance of Gammaproteobacteria increased sharply from 11% to 60%, while the relative abundance of Betaproteobacteria decreased from 57% to 19% (Figure 3.8). In spite of the shift at the class level, the phyla of Proteobacteria dominated both seed and Fe+2-fed cultures. Xanthomonas axonopodis and Thermomonas sp. were the dominant genera with high relative abundances of 36% and 16%, respectively. Xanthomonas sp. have been reported as one of common denitrifying species of Gammaproteobacteria in wastewater treatment plant, soil, and lab-scale fluidized bed reactor (Wanner et al., 1990; Finkmann et al., 2000; Saddler and Bradbury, 2005; Gentile et al., 2006). Thermomonas sp. also have been commonly isolated from denitrifying habitat (Mergaert et al., 2003). Straub et al. (2004) obtained Thermomonas sp. that are capable of reducing nitrate by oxidizing ferrous iron from sediments. Zhang et al. (2018) reported that Thermomonas sp. was the predominant bacteria in Fe+2-oxidizing denitrifying sludge enriched under high Fe2+ concentrations. It should be noted that sharp increases in the relative abundance of Gammaproteobacteria (e.g., Xanthomonas sp. and Thermomonas sp.) observed in the Fe2+-cell reactor was not observed in the ZVI-enriched cultures, even though the ZVI reactors contained Fe2+ ions due to the anaerobic corrosion of ZVI. This result suggests that the Fe2+-supported growth may not be significant in ZVI-cell systems. Conversely, denitrification by cathodic hydrogen may be the dominant reaction in microbial-mediated ZVI-supported nitrate reduction process.

3.3.3 Principal Component Analysis Principal component analysis (PCA) is selected due to its advantages which process the abundant and complex data into simpler sets with smaller number of variables, and visualize the data in more understandable way at a glance (Nicholson et al., 1999; Ringner, 2008). PCA was conducted using the relative abundance data (obtained from Krona chart) to examine the similarity among four treatment groups. A total of 207 sequenced taxonomic groups were identified from 4 different samples, and the sequences, which did not exist in at least one reactors were deleted for the PCA; as a result, 147 taxonomic groups were used for the analysis. Two principal components (PCs) accounted for 74% of the total variation. The first principal component (PC1) explained 48% of total variation and the second principal component (PC2) accounted 26% of the total variation. Figure 3.9 compares the PCA results of 4 treatment groups. The PCA results show that there was a significant shift in the microbial communities in the ZVI- supported cultures compared to the seed cultures (Figure 3.9). The microbial composition of H2-fed cultures was similar to that of ZVI-supported cultures, suggesting that even though the hydrogen sources are different between two cultures, the same populations of bacteria may be involved in denitrification. On the other hand, microbial composition data of the Fe2+-fed cultures were shifted further away from that from both ZVI-cell and H2-cell reactors, indicating that the activity of ferrous- oxidizing bacteria may not be significant in microbial-ZVI systems. These results suggest that the cathodic hydrogen derived from iron corrosion is the primary electron donor for the denitrification in the ZVI-supported microbial systems.

Table 3.1. The electron donors and carbon sources of the batch experiments

e- donor C Source

- H -utilizing Denitrification HCO 2 H2 3

2+ - 2+ HCO Fe - oxidizing Denitrification Fe 3

- ZVI as a precursor of HCO ZVI w/ Cell 2+ 3 H , and Fe 2

1.2 ZVI only

1 Cell only Fe2+-cell 0.8 H2+cell ZVI + cell 0.6 C/Co 0.4

0.2

0 0 1 2 3 4 5 Days

Figure 3.1. Nitrate removals in batch reactors under the different conditions.

50 Nitrate

10 Concentration (mg/L as N) as (mg/L Concentration

0 0 1 2 3 4 5 6 7 8 Days

Figure 3.2. Nitrate removals in H2-fed microbial culture.

50 Nitrate

10 Concentration (mg/L as N) as (mg/L Concentration

0 0 1 2 3 4 5 6 7 8 9 Days

Figure 3.3. Nitrate removals in Fe2+-fed microbial culture.

50 Nitrate Ammonia 40

10 Concentration (mg/L as N) as (mg/L Concentration

0 0 1 2 3 4 5 6 7 Days

Figure 3.4. Nitrate removal in ZVI-mediated microbial culture.

Figure 3.5. Krona graphs of microorganisms content in seed culture identified by 16S rRNA gene sequencing.

Figure 3.6. Krona graphs of microorganisms content in ZVI-supported reactor culture identified by 16S rRNA gene sequencing.

Figure 3.7. Krona graphs of microorganisms content in H2-fed reactor culture identified by 16S rRNA gene sequencing.

Figure 3.8. Krona graphs of microorganisms content in Fe2+-fed reactor culture identified by 16S rRNA gene sequencing.

0.5

0 PC2

Fe2+-fed -0.5 Seed culture H2-fed ZVI+cell -1 -1 -0.5 0 0.5 1 PC1

Figure 3.9. The principal component analysis (PCA) plot.

Chapter 4

EFFECT OF LOW TEMPERATURE ON ABIOTIC AND BIOTIC NITRATE REDUCTION BY ZERO-VALENT IRON

Abstract

- The effects of low temperatures on abiotic and biotic nitrate (NO3 ) reduction by zero-valent iron (ZVI) were examined at temperatures below 25 ℃. The extent and rate of nitrate removal in batch ZVI reactors were determined in the presence and absence of the microorganisms at 3.5, 10, 17, and 25 ℃. Under anoxic conditions,

+ recovered as ammonium ions (NH4 ) at both temperatures. The temperature-dependent abiotic reduction rates enabled us to calculate the activation energy (Ea) from the Arrhenius relationship, which was 50 kJ/mol. Nitrate in the ZVI-cell reactors was completely removed within 1 – 2 days at 25 and 10 ℃, and 67% of reduction was

+ achieved at 3.5 ℃. Only 17 – 23% of the reduced nitrate was recovered as NH4 in the ZVI-cell reactor. Soluble iron concentrations (Fe2+ and Fe3+) in the ZVI reactors were also measured as the indicators of anaerobic corrosion. The concentration of soluble iron in ZVI-cell reactors were 1.7 times higher than that in ZVI-only reactors at 25 oC, suggesting that the enhanced nitrate reduction in the ZVI-cell reactors may be partly due to increased redox activity (i.e., corrosion) on iron surfaces. Anaerobic corrosion of ZVI was also temperature-dependent as substantially lower concentrations of

corrosion product were detected at lower incubation temperatures; however, microbially induced corrosion (MIC) of ZVI was much less impacted at lower temperatures than abiotic ZVI corrosion. This study demonstrated that ZVI-supported microbial process (i.e., denitrification) is not only more sustainable at lower temperatures, but it becomes more dominant reaction for nitrate removal in microbial- ZVI systems at low temperatures.

4.1 Introduction

- Waters contaminated with nitrate (NO3 ) are commonly treated by physio- chemical processes such as ion exchange or reverse osmosis. However, these conventional processes are costly and difficult to operate, and generate concentrated waste streams that may cause further disposal problems (Ergas and Reuss, 2001; Shrimali and Singh, 2001). As an alternative to these conventional process, nitrate reduction by zero-valent iron (ZVI) has been demonstrated as an effective chemical process for nitrate removal (Huang et al., 1998; Tratnyek et al., 2003; Ahn et al., 2008; Zhu and Getting, 2012; Suzuki et al., 2013; Liu et al., 2013). In the absence of oxygen, nitrate reduction by ZVI is thermodynamically favorable with ammonia and ferrous iron as the major products according to the following reaction (Cheng et al., 1997):

0 - + + 2+ 4Fe + NO3 + 10H → NH4 + 4Fe 3H2O (4.1)

It has been reported that the rate of nitrate reduction by ZVI is strongly influenced by acidic conditions (Choe et al., 2004; Huang and Zhang, 2004) as various acidic species such as strong acids, acetic acid, and CO2 gas have been used to accelerate the reaction rate (Huang et al., 1998; Cheng et al., 1997; Su and Puls, 2004; Ruangchainikom et al., 2006; Ahn et al., 2012). Although the application of acidic conditions are effective in laboratory studies, lowering the solution pH may not be

economically practical and may have other adverse secondary impacts on the treated water quality. In addition to abiotic reduction by ZVI, nitrate can also be reduced to nitrogen gas (N2) by ZVI in the presence of denitrifying bacteria (Till et al., 1998; Mansell and Schroeder, 2002; Rocca et al., 2007; Shin and Cha, 2008; An et al., 2009; Peng et al.,

2015). Cathodic hydrogen (H2) is generated from anaerobic oxidation of ZVI coupled with the reduction of proton according to the following equation (Daniels et al., 1987; Häring and Conrad, 1991; Smith et al., 1994; Till et al., 1998):

0 2+ - Fe + 2H2O → H2 + Fe + 2OH (4.2) This hydrogen gas is a thermodynamically favorable electron donor for nitrate- respiring bacteria (Kurt et al., 1987). Microbially induced corrosion (MIC) is a well-known process that relates the activity of microorganisms and anaerobic corrosion of iron in the environment (Stott, 1993; Xu et al., 2013; Enning and Garrelfs, 2014). The MIC process promotes corrosion of iron or carbon steel by the removal and oxidation of cathodic hydrogen through bacterial enzymatic activities (Gains, 1910; Stott, 1993). The consumption of cathodic hydrogen by hydrogenophilic bacteria accelerates anodic ZVI oxidation (Lino et al., 2015), which naturally results in the corrosion of iron. This type of biocorrosion has been extensively studied, especially for sulfate- and nitrate-reducing bioreactors (Hamilton, 1985; Dowling et al., 1992; De Windt et al., 2003; Hubert et al., 2005; Mori et al., 2010; Xu et al., 2013). Some denitrifying strains, including Pseudomonas sp. M9 and Shewanell oneidensis MR-1, have been attributed to biocorrosion of the ZVI beads system under nitrate-reducing conditions (Pedersen et al., 1988; De Windt et al., 2003). In the

absence of oxygen, the cell density around the iron particles was shown to increase.

The accelerated consumption of H2 film from the surfaces of iron resulted in an increased solubilization of iron minerals including Fe2+ and Fe3+ from ZVI particles, resulting in 1.28 times more total iron solubilization than that from the abiotic control test (De Windt et al., 2003). Furthermore, the presence of microorganisms stimulated both iron corrosion and nitrate degradation kinetics at the standard conditions, while ZVI-only system did not (Kaesche, 2003). Nitrate removal rate by ZVI particles has been shown to be temperature- dependent in many previous studies (Ginner et al., 2004; Ahn et al., 2008). Nitrate and nitrite reduction by Master Builder iron filings over the range of temperature (5 to 50 ℃) were examined in batch reactors under the buffered condition at pH 8.4 (Ginner et al., 2004). The reduction rates of nitrate and nitrite increased as temperature increased in the buffered-mineral solution. Moreover, the faster degradation of nitrate was observed in the presence of denitrifying bacteria, Paracoccus denitrificans, compared to the abiotic nitrate reduction by iron particles at the same temperatures. Ahn and his colleagues (2008) reported that the increase in solution temperature up to 75 ℃ accelerated the rate of abiotic nitrate reduction both in batch and column reactors. Most of the reported studies on abiotic and biotic nitrate reduction by ZVI were conducted at the ambient temperature and above. There is no study evaluating nitrate reduction reactions at low temperatures below 25 ℃, especially with microorganisms. It will be beneficial to comprehend kinetic information on both abiotic and biotic nitrate reduction in the presence of ZVI at low temperatures would be valuable for the design and operation of field-scale ZVI systems, especially for winter operations.

In this study, nitrate reductions by ZVI was examined at low temperatures in the presence and absence of microorganisms. Emphasis was on investigating the role of microorganisms and effect of temperature on nitrate removal efficiency and ammonia production. The study also examined microbially induced corrosion process at the ZVI surfaces to elucidate the interactions and synergies between ZVI and microorganisms in microbial-ZVI systems.

4.2 Materials and Methods

4.2.1 Chemicals and Microorganisms Zero-valent iron (ZVI) granules were purchased from Peerless Metal Powders

Inc. (Detroit, MI), and sieved with 18-20 mesh. Sodium nitrate (NaNO3, 99%) was obtained from Sigma-Aldrich (St. Louis, MO). The culture medium contained 300 mg/L of NaHCO3 as the inorganic carbon source for autotrophic microorganisms, 300 mg/L of KH2PO4, 100 mg/L of MgCl2‧6H2O, 50 mg/L of MgSO4‧7H2O, 25 mg/L of

CaCl2‧2H2O, and 1 mL of trace mineral solution. The composition of trace mineral solution was as follows: 450 mg/L FeCl2‧4H2O, 190 mg/L CoCl2‧6H2O, 100 mg/L

MnSO4‧7H2O, 52 mg/L ZnCl2, 36 mg/L Na2MoO4‧2H2O, 30 mg/L H3BO3, 29 mg/L

CuCl2‧2H2O, and 24 mg/L NiCl2‧6H2O. Seed cultures for the batch reduction experiments were obtained from Elkton Wastewater Treatment Plant (Elkton, MD). This plant is a biological nutrients removal plant designed for simultaneous nitrification/denitrification process. Thus, well- established denitrifying cultures were obtained for our tests without any acclimation or enrichment.

4.2.2 Batch Reduction Experiments Batch reduction experiments were conducted using 250 mL amber bottles. Each biotic bottle contained 100 mL of medium, 5 g of ZVI, and 1,000 mg/L TSS of seed cultures. Abiotic bottles contained the same amount of medium and ZVI but were not seeded with activated cultures. Both biotic and abiotic bottles were purged with N2 for at least 10 minutes to remove residual dissolved oxygen and tightly sealed with screw-top MininertTM caps (Sigma-Aldrich, MO). Batch reduction tests were conducted at four different temperatures: 3.5, 10, 17, and 25 ℃. For the 3.5 ℃ study, the experiments were conducted in the temperature-controlled room. The test bottles were continuously shaken on a platform shaker (New Brunswick Scientific, Edison, NJ) at 150 rpm. For the 10 and 17 ℃ studies, a shaking water bath (Thermo Scientific, Asheville, NC) connected to a Polystat temperature controller (Cole-parmer, Vernon Hills, IL) was used. For the 25 ℃ study, the test bottles were incubated in a temperature-controlled chamber equipped with a platform shaker. All bottles were continuously shaken in a horizontal position at 150 rpm. All reduction experiments were conducted in triplicate and initial nitrate concentration in each bottle was 10 – 12 mg-N/L. At selected time intervals, the samples from the test bottles were collected with gas-tight syringe, and immediately filtered through a 0.2 µm membrane filter (Fisher Scientific, Hampton, NH) for the chemical analyses.

4.2.3 Analytical Procedures Nitrates were analyzed using a Dionex ICS-1000 ion chromatography (IC) (Dionex, Sunnyvale, CA) equipped with a Dionex AERS 500 suppressor, and Dionex IonPac AS23 capillary column in combination with the Dionex IonPac AG23 guard

column. Separation of samples was achieved at 30 ℃ using an eluent solution mixture of 4.5 mM of Na2CO3 and 0.8 mM of NaHCO3 and injection volume was 25 μL. Ammonia ion and total iron were analyzed with the Hach spectrophotometer DR 5000 (Hach, Loveland, CO) using the salicylate and FerroVer methods, respectively.

4.3 Results and Discussion

4.3.1 Effects of Temperature on Nitrate Reduction by ZVI Batch nitrate reduction by ZVI under various temperatures are presented in Figure 4.1. The nitrate reduction by ZVI under unbuffered condition was slow and incomplete. Only 60% of nitrate was removed after 6 days at 25 ℃. Figure 4.1(a) shows the rate and extent of nitrate reduction further decreased at lower temperatures. Only 17.3% of nitrate was reduced in the ZVI reactor system at 3.5 ℃ after 6 days, while 34.6, 46.3, and 61.3% of nitrate was removed at 10, 17, and 25 ℃, respectively, during the same 6-day period. The pH of the solution increased from 7.6 to 8.9 in the most of the cases. Figure 4.1(b) shows the accumulated concentrations of ammonium

- ions produced in ZVI only reactors at the various temperatures. The reduction of NO3

+ was almost entirely to NH4 at 3.5, 10, 17, and 25 ℃. This transformation of nitrate to ammonium ions by ZVI is consistent with previously reported studies (Huang et al.,

1998; Westerhoff, 2003; Huang and Zhang, 2004; Su and Puls, 2004).

The pseudo-first-order rate constants (kobs) for nitrate reduction by ZVI were calculated to be 0.032 (R2 = 0.88), 0.068 (R2 = 0.97), 0.10 (R2 = 0.99), and 0.16 day-1 (R2 = 0.99) at 3.5, 10, 17, and 25 ℃, respectively (Table 4.1 and Figure 4.2). The results clearly indicate that the rate of nitrate reduction by ZVI is strongly temperature

dependent, suggesting ZVI-based nitrate removal processes may be ineffective under low temperature conditions. Arrhenius equation has been commonly applied to the experimental data to calculate activation energy (Ea) for nitrate reduction by ZVI (Ginner et al., 2004; Ahn et al., 2008):

k = A exp (-Ea/RT) (4.3) where Ea is the activation energy (kJ/mol), A is the frequency factor for the reaction, and R is the universal gas constant (8.314 J/K‧mol). The Ea value calculated from the slope of the linearized form of the Arrhenius equation (Figure 4.3) was 50 kJ/mol.

Since the calculated Ea value was obtained under unbuffered conditions, the value was substantially greater than the reported values of 28.2 kJ/mol (Ahn et al., 2008) and 21.7 kJ/mol (Ginner et al., 2004) for nitrate reduction by ZVI in buffered systems. This result indicates that unbuffered ZVI reduction of nitrate is more sensitive to temperature shifts and may be less effective at low temperatures than buffered processes.

4.3.2 Effects of Temperature on Nitrate Reduction by ZVI and Microorganisms The inhibitory effect of low temperature on ZVI reduction of nitrate is more moderate in the presence of denitrifying cells (Figure 4.4(a)). At 25 ℃, nitrate in the batch reactor containing ZVI and cells was completely removed within one day. The nitrate removal in ZVI-cell bottle was slightly slower at 10 ℃ as about 7% of the initial nitrate was remaining after 1-day incubation. The remaining nitrate was completely removed in day 2. The 25 oC ZVI-cell reactor was spiked with nitrate on day 1 and a similar removal rate was observed after the re-spiking (Figure 4.4(a)). Nitrate removal rate in the ZVI-cell reactor decreased as the incubation temperature

was further decreased to 3.5 ℃; however, the presence of microbial cell still resulted in the removal of 60% of initial nitrate concentration in 6 days, which is substantially greater than that observed in the abiotic ZVI reactor. The ZVI-only test resulted in only 9% removal in 6 days at 3.5 ℃. Moreover, the presence of denitrifying mixed cultures in ZVI reactors resulted in less production of ammonium ions from nitrate. At 25 ℃, less than 21% of

- + removed NO3 was recovered as NH4 (Figure 4.4(b)). Similar recovery rate (23%) was observed after re-spiking of 25 ℃ reactor with nitrate. The percent recovery of ammonium ions further decreased at 3.5 ℃ as only 17% of removed nitrate was recovered as ammonium ions. Since the abiotic tests showed that the majority of removed nitrate were transformed to ammonium ions, the presence of ammonium ions in microbial-ZVI reactors was most likely due to abiotic nitrate reduction by ZVI. Greater nitrate removal in microbial-ZVI reactors suggests that microbially-mediated reduction may be responsible for additional nitrate removal. We were not able to establish complete nitrogen balance in microbial-ZVI reactors because N2 gas and

N2O, the well-known product and intermediates of microbial denitrification, were not measured in this study. Nevertheless, greater nitrate removal and lower ammonia production clearly demonstrated the beneficial role of denitrifying cultures in ZVI system for nitrate reduction even at low temperatures.

4.3.3 Enhanced Denitrification by Microbial Induced Corrosion

Under anoxic conditions, a H2 film forms at the ZVI surface that inhibits the electron flow from the surface, thus protecting the metal surface from further corrosion (Reardon, 1995; Mackenzie et al., 1999). One possible reason for more efficient nitrate reduction in microbial-ZVI system than ZVI only system (even at low

temperature) may be microbially induced corrosion (MIC). Many microorganisms including denitrifying bacteria can grow on corrosion products of ZVI such as hydrogen gas (Pedersen et al., 1988; Kielemoes et al., 2000; De Windt et al., 2003; Biswas and Bose, 2005; Hubert et al., 2005; Xu et al., 2013). Hydrogen consumption by denitrifying bacteria growing at ZVI surface may cause the removal of the protective H2 layer, which can accelerate the electron transfer. To evaluate the role of microorganisms on the corrosion rate of ZVI, corrosion products from iron granules, ferrous (Fe2+) and ferric (Fe3+) ions were quantified. Figure 4.5(a) shows that the concentration of soluble iron in the ZVI only reactor was 69 mg/L at 25 ℃. The soluble iron concentration was increased to 114 mg/L in the presence of bacteria at the same incubation temperature of 25 ℃ (Figure 4.5(b)), indicating more corrosion occurred in the presence of bacteria. Abiotic corrosion process accounted for about 60% of soluble iron in ZVI-cell reactors. Anaerobic corrosion of ZVI was also temperature-dependent as substantially lower concentrations of corrosion product were detected at lower incubation temperatures (Figure 4.5). Soluble iron concentrations decreased to 24 mg/L at 10 ℃ and 17 mg/L at 3.5 ℃ in the ZVI-only reactors (Figure 4.5(a)). The concentrations of corrosion products also decreased in ZVI-cell reactors, but relatively high iron concentrations of 88 mg/L and 71 mg/L were still detected at 10 and 3.5 ℃, respectively, indicating corrosion activities may be sustained at lower temperatures in the presence of bacteria. Furthermore, Figure 4.5(b) shows that at lower temperatures, microbial process becomes more significant mechanisms for the corrosion of ZVI. At 3.5 ℃, abiotic dissolution of iron accounted for only about 23.9% of released iron into solution of

ZVI-cell reactor compared to 60% abiotic portion observed at 25 ℃. These results suggest that microbial process (i.e., denitrification) is more dominant reaction for nitrate removal in ZVI systems at low temperatures. Considering the nitrate reduction rate and the relative amount of iron corrosion

- products, it can be concluded that enhanced NO3 reduction in ZVI reactors is due to microorganisms stimulating iron corrosion for acquiring electron donors. Figure 4.6 illustrates a schematic of this ZVI-supported nitrate reduction process. The microbial process is preferred over the abiotic reduction process because it is not only faster, but also results in lower abiotic ammonia production, which requires further treatment. We have thus far demonstrated that ZVI-supported denitrification may be practical and sustainable applications to remove nitrate at lower temperature conditions. However, further studies on biofilm formation and microbial ecology may be valuable to better understand the interactions and synergies between ZVI and microorganisms at the ZVI surfaces.

4.4 Conclusion The experimental results showed that the ZVI technology can be applied at low temperatures to reduce nitrate. Nitrate reduction in ZVI-only and ZVI-cell reactors were both temperature dependent as nitrate reduction rates increase with increases in temperature. For abiotic nitrate reduction by ZVI in unbuffered solutions, the calculated activation energy (Ea) was 50 kJ/mol. The addition of denitrifying bacteria to ZVI granules accelerated nitrate reduction and produced less ammonium ions. Soluble iron concentrations (Fe2+ and Fe3+) in the ZVI reactors were also measured as the indicators of anaerobic corrosion. In ZVI-cell reactors, the detected soluble iron concentrations were 1.7, times higher

than that in ZVI-only reactors at 25 oC, suggesting that the enhanced nitrate reduction in the ZVI-cell reactors may be partly due to increased redox activity (i.e., corrosion) on iron surfaces. Anaerobic corrosion of ZVI was also temperature-dependent as substantially lower concentrations of corrosion product were detected at lower incubation temperatures; however, microbially induced corrosion (MIC) of ZVI was much less impacted at lower temperatures than abiotic ZVI corrosion. This study demonstrated that ZVI-supported microbial process (i.e., denitrification) is not only more sustainable at lower temperatures but it becomes more dominant reaction for nitrate removal in microbial-ZVI systems at low temperatures.

-1 Table 4.1. Estimated the effects of temperature on kobs (day ) for abiotic nitrate reduction by ZVI

Temperature (°C) 3.5 10 17 25

-1 -2 -2 -1 -1 kobs (day ) 3.2 × 10 6.8 × 10 1.0 × 10 1.6 × 10

16 (a) 3.5 °C 14 10 °C 17 °C 12 25 °C 10

2 Nitrate Concentration (mg/L as N) as (mg/L Concentration Nitrate 0 0 1 2 3 4 5 6 Time (day) 12 (b) 25 °C 10 17 °C 10 °C 8 3.5 °C

2 Ammonia Concentration (mg/L as N) as (mg/L Concentration Ammonia 0 0 1 2 3 4 5 6 Time (Day) Figure 4.1. Nitrogen concentrations of the unbuffered ZVI batch reactors operated o - + at 25, 17, 10, and 3.5 C: (a) NO3 , and (b) NH4 concentration.

t vs. ln(C/Co)

-0.2 y = -0.0319x - 0.0143

-0.4 3.5 °C y = -0.0684x + 0.0142

10 °C ln(C/Co) -0.6 y = -0.1015x + 0.0062 17 °C -0.8 25 °C y = -0.1595x + 0.0246 -1 0 1 2 3 4 5 6 Time (day)

Figure 4.2. The estimation of nitrate reduction rate by ZVI under unbuffered abiotic condition at 3.5, 10, 17, and 25 oC.

-0.5

-1

-1.5

-2 lnk

-2.5

-3 y = -6019.1x + 18.428 -3.5 R² = 0.9695

-4 0.0032 0.0034 0.0036 0.0038 1/T

Figure 4.3. Arrhenius plots of nitrate removal by ZVI in batch reactors.

16 (a) 14 25 °C 10 °C 12 3.5 °C 10

2 Nitrate Concentration (mg/L as N) as (mg/L Concentration Nitrate

0 0 1 2 3 4 5 6 Time (Day) 12 (b) 25 °C 10 10 °C 3.5 °C 8

2 Ammonia Concentration (mg/L as N) as (mg/L Concentration Ammonia 0 0 1 2 3 4 5 6 Time (Day)

Figure 4.4. Nitrogen concentrations of the unbuffered microbial-ZVI batch reactors o - + operated at 25, 10, and 3.5 C: (a) NO3 , and (b) NH4 concentration.

200 (a) (b) 180 160 ZVI only ZVI with bacteria 140 120 100 80 60 40 Iron Concentration (mg/L) Concentration Iron 20 0 25 °C 17 °C 10 °C 3.5 °C 25 °C 10 °C 3.5 °C Temperature

Figure 4.5. Soluble iron concentration including ferrous (Fe2+) and ferric (Fe3+) iron from batch reactors after 6 days. (a) reactor with ZVI only; (b) reactor with ZVI and cell.

Anoxic

(a) Abiotic (b) Biotic (slow) - (fast) NO3 N2 Fe2+/Fe3+ Denitrifying bacteria + - + NH4 NO3 2H H2

e- e- Fe (0)

- - 5 H + 2 NO  N + 4 H O + 2OH 2 3 2 2

Figure 4.6. Schematic illustration of possible pathway of abiotic and biotic nitrate reduction by zero-valent iron (ZVI).

Chapter 5

NITROGEN REMOVAL BY MICROBIALLY-COLONIZED IRON GRANULES: ANAMMOX-LIKE PROCESS

Abstract Anaerobic ammonia oxidation (anammox) is a promising nitrogen removal process that may occur in zero-valent iron (ZVI)-supported denitrification process. Electron donor (ammonia) and acceptor (nitrite) for the anammox reaction are typically present in microbial-ZVI systems. In this study, it was hypothesized that ZVI granules could serve as support media for the enrichment of anammox biofilm. The feasibility of ZVI-supported anammox-like process was demonstrated in a preliminary batch study. An anaerobic fluidized bioreactor (AFBR), using ZVI granules as the solid support materials for the biomass was operated for 50 days to study the potential colonization and stable biofilm formation of ZVI surfaces by anammox-performing cultures. During the 50-day experimental period, the nitrate was completely removed with minimal production of ammonium ions. The confocal images showed that microbial colonies were formed not only at the ZVI surface, but also within the crevices of ZVI particles. Scanning electron micrograph of the ZVI granule from the AFBR showed that the ZVI surface was completely covered by the deposition of microorganisms and minerals. Genomic analysis of biofilm on the ZVI surface was conducted to examine the diversity and abundance of microbial communities in the ZVI-supported biofilm. Even though the relative abundance of Planctomycetes in the ZVI culture was small, the detection of Planctomycetes combined with the chemical

data supporting anammox-like process suggested that low ammonia accumulation in the AFBR may be due to anammox activity.

5.1 Introduction Denitrification is a widely present microbial process for nitrate removal in the environment. Due to the high processing rates, and its innocuous product, nitrogen gas

(N2), biological denitrification is considered as very effective nitrate treatment method (Payne, 1976; Matéju et al., 1992; Oh et al., 2001; Steingruber et al., 2001). Denitrifying bacteria receive electrons from electron donors such as hydrogen gas

2+ (H2), ferrous iron (Fe ), sulfur element (S), and organic compounds, and transfer

- electrons to the terminal electron acceptor, NO3 (Knowles, 1982; Straub et al., 1996; Till et al., 1998; Heylen et al., 2006; van Rijn et al., 2006). This anaerobic respiration process is performed through electron transfer chain (ETC) in bacterial membrane (Bertero et al., 2003). It has been shown that zero-valent iron (ZVI) sustains autotrophic denitrification as a continuous source of electron donors (Till et al., 1998; Shin and Cha, 2008). Hydrogen gas is a major electron donor generated from the anaerobic corrosion of ZVI in water according to the following equation (Agrawal and Tratnyek, 1995; Till et al., 1998):

0 2+ - Fe + 2 H2O  Fe + H2 + 2 OH (5.1)

Hydrogen is thermodynamically favorable electron donors for nitrate reduction and is readily utilized by hydrogenophilic denitrifiers for autotrophic denitrification (Thauer et al., 1977; Shin and Cha, 2008; Lee et al., 2010; Blodau, 2011):

- - 2 NO3 + 5 H2  N2 + 4 H2O + 2 OH (5.2)

In addition to supporting biotic nitrate reduction by providing electron donors to denitrifying bacteria, ZVI can also chemically reduce nitrate to ammonia according to the following reaction (Cheng et al., 1997; Till et al., 1998):

- 0 + + 2+ NO3 + 4 Fe + 10 H  NH4 + 4 Fe + 3 H2O (5.3) The above reduction reaction is thermodynamically favorable (Go = -460 kJ/mol), but ammonium production from abiotic nitrate reduction limits the environmental application of ZVI. Anaerobic ammonia oxidation (anammox) is a promising process that may occur in ZVI-supported denitrification. Anammox bacteria gain energy by oxidizing

+ - ammonium ion (NH4 ) with transferring the electrons to nitrite (NO2 ) generating innocuous nitrogen gas (N2) (van de Graaf et al., 1996):

+ - NH4 + NO2  N2 + 2 H2O (5.4)

Anammox reaction was firstly observed in a laboratory-scale fluidized-bed reactor

- (FBR) with NO3 as a sole electron acceptor (Mulder et al., 1995). The authors observed ammonium losses in the denitrifying FBR containing sand carriers coated with denitrifying biofilms. Subsequent batch test with the FBR cultures showed that

- nitrite (NO2 ) could serve as an electron acceptor for the anammox process instead of nitrate, and anammox bacteria can be autotrophically grown in synthetic inorganic medium (van de Graaf et al., 1996; Strous et al., 1999). Also, the phylogenetic analysis of anammox cultures identified the responsible bacteria as the phylum of Planctomycetes (Strous et al., 1999). Since the first full-scale anammox process was put into operation in treatment plants in 2002 (Jetten et al., 2001; Mulder et al., 1995), many municipal and wastewater treatment plants have included the anammox process

for the efficient and cost-effective removal of nitrogen (van Dongen et al., 2001; Schmidt et al., 2003; Abma et al., 2007; Kartal et al., 2010; Lackner et al., 2014). It has been reported that the anammox process not only saves up to 65% of aeration energy cost, but also reduces up to 100% of external carbon supply and 50% of alkalinity requirements as compared to conventional nitrification-denitrification process in the wastewater treatment plant (York River treatment plant, 2015). We hypothesized that ZVI granules could serve as support media for the enrichment of anammox biofilm. The presence of anammox bacteria in the ZVI- supported denitrifying system could then decrease the generation of ammonia in microbial-ZVI systems as anammox bacteria can oxidize ammonium with nitrate under the anaerobic and autotrophic conditions (Mulder et al., 1995; van de Graaf et al., 1996; Strous et al., 1999). Both of electron donor (ammonia) and acceptor (nitrite) for the anammox reaction are produced during ZVI-supported denitrification process (Payne, 1976; Till et al., 1998; Shin and Cha, 2008), and thus, ammonia as a product of abiotic nitrate reduction by ZVI (Till et al., 1998; Shin and Cha, 2008). Consequently, more desirable reduction of nitrate into innocuous nitrogen gas is expected to occur due to the formation of anammox biofilm on ZVI surfaces. Generally, microorganisms have two forms of growth in water: planktonic microorganisms, which freely move in bulk solution, and sessile microorganisms, which attach to a substratum, such as the surface of metals, living tissues, plastics, etc. (Heukelekian and Heller, 1940; Dunne, 2002). The formation of biofilm on the solid surfaces is initiated from the cell-surface interactions in response to specific environmental signals such as nutrient availability or protection from inhibitory compounds (O’Toole et al., 2000). When bacteria switches to the biofilm mode of

growth, it is, therefore, beneficial for bacteria as they are able to obtain nutrients in oligotrophic conditions and are sheltered from harmful factors in the environment (Tuson and Wiedel, 2013). Bacterial colonization of zero-valent metal surfaces have been widely reported (Beech and Sunner, 2004). Especially, there have been many studies on the microbial colonization on the ZVI surfaces by sulfate-reducing bacteria

2- (SRB), which also uses cathodic H2 gas as electron donor to reduce sulfate (SO4 ) to hydrogen sulfide (H2S) under the anaerobic condition (Videla, 2000; Beech and Campbell, 2008; Celis et al., 2009). The objective of the research reported in this chapter is to study the potential colonization of ZVI surfaces by anammox-performing cultures. The feasibility of ZVI- supported anammox process was demonstrated in a preliminary batch study. The anaerobic fluidized bed bioreactor was operated for 50 days to promote colonization and stable biofilm formation on the ZVI surface. Genomic analysis of biofilm on the ZVI surface was conducted to examine the diversity and abundance of microbial communities in the ZVI-supported biofilm.

5.2 Materials and Methods

5.2.1 Batch Reduction Tests with Anammox Mixed Cultures

Anammox seed cultures for batch reduction experiments were obtained from HRSD York River wastewater treatment plant (Seaford, VA). This plant applies partial nitritation by ammonia oxidizing bacteria (deammonification) combined with anammox processes for nitrogen removal. Thus, their mixed cultures were expected to include anammox bacteria.

ZVI granules were purchased from Peerless (Detroit, MI), and sieved with 18-

20 mesh. Sodium nitrate (NaNO3, 99%) was obtained from Sigma-Aldrich (St. Louis,

MO). The culture medium was contained 300 mg/L of NaHCO3, 300 mg/L of

Na2MoO4‧2H2O, 30 mg/L of H3BO3, 29 mg/L of CuCl2‧2H2O, and 24 mg/L of

NiCl2‧6H2O. To maintain an optimal physiological pH between 7.2 and 7.6, 0.1 M of HEPES was added (Good et al., 1966; Shin and Cha, 2008; Saleh-Lakha et al, 2009). Batch nitrate reduction experiments were conducted using 250 mL amber bottles. Each biotic bottle contained 100 mL of medium, 30 g of ZVI, and 1,000 mg/L

TSS of seed cultures. All bottles were purged with N2 for at least 10 minutes to remove residual dissolved oxygen and tightly sealed with screw-top MininertTM caps (Sigma-Aldrich, MO). Batch reduction tests were conducted at room temperature. The test bottles were continuously shaken on a platform shaker (New Brunswick Scientific, Edison, NJ) at 150 rpm.

- The bottles initially contained 40 mg/L NO3 -N and nitrate was replenished after complete consumption. Nitrates were analyzed using a Dionex ICS-1000 ion chromatography (IC) (Dionex, Sunnyvale, CA) equipped with a Dionex AERS 500 suppressor, and Dionex IonPac AS23 capillary column in combination with the Dionex IonPac AG23 guard column. Separation of samples was achieved at 30 ℃ using an eluent solution mixture of 4.5 mM of Na2CO3 and 0.8 mM of NaHCO3 and injection volume was 25 μL. Ammonia ions were analyzed with the Hach spectrophotometer DR 5000 (Hach, Loveland, CO) using the salicylate methods.

5.2.2 Denitrification in Anaerobic Fluidized Bioreactor Anaerobic fluidized bioreactor (AFBR), using ZVI granules as the solid support materials for the biomass was designed to investigate the biofilm formation on ZVI surfaces. Seed cultures for the AFBR were obtained from Elkton Wastewater Treatment Plant (Elkton, MD). This plant is a biological nutrients removal plant designed for simultaneous nitrification/denitrification process. Thus, the plant mixed cultures were used in our tests without any acclimation or enrichment as it was expected to include well-established denitrifying communities (EVOQUA, 2014).

The culture medium contained 300 mg/L of NaHCO3 as an inorganic carbon source for autotrophic microorganisms, 300 mg/L of KH2PO4, 100 mg/L of

MgCl2‧6H2O, 50 mg/L of MgSO4‧7H2O, 25 mg/L of CaCl2‧2H2O, and 1 mL of trace mineral solution. The composition of trace mineral solution was as follows: 450 mg/L

FeCl2‧4H2O, 190 mg/L CoCl2‧6H2O, 100 mg/L MnSO4‧7H2O, 52 mg/L ZnCl2, 36 mg/L Na2MoO4‧2H2O, 30 mg/L H3BO3, 29 mg/L CuCl2‧2H2O, and 24 mg/L

NiCl2‧6H2O. The pH of the system was maintained around 7 by 0.1 M HEPES (4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid)-buffered nutrient media (Shin and Cha, 2008). An AFBR column was constructed using an acrylic pipe with a height of 50 cm, an internal diameter of 2.54 cm (Figure 5.1). The bottom of the reactor was filled with glass beads (OD = 3 mm, Fisher Scientific, Hampton, NH) for uniform dispersion of the up-flow solution (Smedt and Wierenga, 1984). The AFBR contained 30 g of ZVI granules, which was sieved with size 8/50 (same as 0.093 inches). The bottom and top of the AFBR was connected to a separate reservoir, and a peristaltic pump (Cole-Parmer, Vernon Hills, IL) was used to continuously deliver the feed solution from the reservoir to the bottom of the AFBR. The working volume was

around 1 L, and appropriate amounts of sodium nitrate (NaNO3) was added to the reservoir every 3 – 5 days to re-spike the medium with nitrate. The whole system was purged with N2 for at least 10 minutes to remove residual dissolved oxygen to maintain anoxic condition in the AFBR system. The reservoir also included a gas vent to prevent any pressure build up from the gases generated from denitrification and anammox processes. Nitrate was analyzed by using a Dionex ICS-1000 ion chromatography (IC) (Dionex, Sunnyvale, CA), and ammonium ions were analyzed with the Hach spectrophotometer DR 5000 (Hach, Loveland, CO) using the salicylate methods.

5.2.3 Surface Study of Zero-valent Iron It was hypothesized that biofilms would be formed on the surface of iron particles during the operation of AFBR. ZVI particles for the surface studies were collected from the bioreactor, and treated according to the required procedures.

5.2.3.1 Confocal Microscopy Microscopic images were obtained with LSM 880 confocal scanning microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with argon (Ar) laser as the light source. To acquire the distributed or colonized bacterial images on the surface, ZVI particles were incubated in 5 uM of SYTO green-fluorescent nucleic acid stain (Thermo Fisher Scientific, Waltham, MA) diluted with phosphate-buffered saline (PBS) solution for 10 minutes. The pretreated iron particles were transferred to NuncTM Lab-TekTM II chambered coverglass (Thermo Scientific, Waltham, MA) for imaging. The image of stained microorganisms and ZVI surface were acquired by using fluorescence mode and reflection mode, respectively.

5.2.3.2 Scanning Electron Microscopy Detailed images of the ZVI surface was obtained from scanning electron microscopy (SEM, Auriga 60 CrosssBeam, Zeiss, Thornwood, NY). A series of pretreatment of the samples were performed to minimize the bacterial cell shrinkage, collapse, and distortion (Gusnard and Kirschner, 1977; Golding et al., 2016). The microorganisms on the ZVI surface were fixed in 2% phosphate-buffered glutaraldehyde (Electrom Microscopy Sciences (EMS), Hatfield, PA) for 12 - 24 hours at 0 - 4 °C, washed with filtered phosphate buffer three times for 5 minutes each. Post staining with 1% osmium tetroxide (OsO4, EMS, Hatfield, PA) was followed by washing with filtered deionized water three times. Then, the dehydration was performed by increasing concentrations of 200 proof anhydrous ethanol (25%, 50%, 75%, 95%, and 100%) diluted with deionized water for 10 - 15 minutes for each concentration. A specimen was finally dehydrated by critical point dryer (CPD, Autosamdri-815B, Tousimis, Rockville, MD) for 1.5 hours. The specimens were coated with platinum by sputter (EM ACE 600, Leica, Buffalo Grove, IL) to add conductivity for imaging.

5.2.4 Identification of Bacterial Community Colonizing Zero-valent Iron Microbial communities established on the ZVI surfaces in denitrifying bioreactor were identified by prokaryotic 16S ribosomal RNA (16S rRNA) gene sequencing. Since 16S rRNA is a valuable phylogenetic marker as the most common “housekeeping gene”, it has been targeted to study bacterial community diversity (Pace, 1997; Janda and Abbott, 2007; Caporaso et al., 2011). ZVI particles from the AFBR were separate from the solution by passing through a 1-mm mesh. Liquid sample were centrifuged at 12,000 rpm for 5 minutes to

collect the suspended biomass, and the supernatant was discarded. Genomic DNA were extracted from both ZVI particles and the pellets with BioReagentsTM SurePrepTM Soil DNA Isolation Kit (Fisher Scientific, Hampton, NH) according the manufacturer’s instructions. The concentration and purity of extracted gDNA were determined using a NanoDrop ND-100 spectrophotometer (Nanodrop Tech, Wilmington, DE). The extracted gDNA was amplified by using the primer set 515F (5’- GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GACTACHVGGGTWTCTAAT- 3’) to amplify variable V4 region among the 16S rRNA gene (Caporaso et al., 2011). The 16S rRNA amplicon was sequenced by Illumina MiSeq sequencing platform at the RTL Genomics (Lubbock, TX). Illumina sequence data were obtained for the paired end reads (2×250), then demultiplexed by the Illumina software. After quality checking and taxonomic identification, the final data were presented on Krona pie graphs, which represents the species diversity per sample (RTL Genomics, 2016). Krona chart is a powerful metagenomic visualization method which composes and displays taxonomic data from the samples (Ondov et al., 2011). A dominant microbial species were identified by comparing the relative abundance of each species.

5.3 Results and Discussion

5.3.1 Anammox Activities in the Presence of Zero-valent Iron: Preliminary Tests In order to evaluate whether anammox process can be established in the ZVI- cell reactor to suppress the ammonia accumulation, a preliminary batch reduction study was conducted with ZVI and mixed cultures containing anammox bacteria. Complete removal of nitrate was observed within 3 days in the batch reactor

containing ZVI and anammox seed bacteria (Figure 5.2) and ammonium ion accumulation in the reactor was less than 8% of the initial nitrate concentration (40 mg/L) (Figure 5.2). In previous batch nitrate reduction studies with non-anammox seed cultures, we observed that about 30% of reduced nitrate was recovered to ammonium ion (Chapter 3). Substantially lower ammonia accumulation observed in this study may be attributed to the presence of anammox bacteria. Repetitive nitrate spiking to replenish the nitrate the batch reactors enabled us to assess whether iron and mixed culture including anammox bacteria can sustain nitrate reduction over time.

5.3.2 Enhanced Nitrate Reduction in Anaerobic Fluidized Bioreactor Nitrate removal by ZVI-supported anammox-like activities was further examined in an anaerobic fluidized bed ZVI bioreactor. The fluidized-bed reactor was chosen to promote good mixing and maximum exposure of ZVI surfaces for biofilm formation. The settling velocity (vs) of ZVI particles was estimated to be 0.4 m/s using Stoke’s equation. Thus, the flow rate was maintained above 500 ml/min to keep the ZVI particles suspended. The AFBR was operated for 50 days (Figure 5.3). Influent nitrate concentrations were decreased to about 10 mg/L N after 2 weeks of operation to avoid high accumulation of ammonia from the abiotic nitrate reduction by ZVI. After each re-spiking, the concentrations of nitrate in the effluent samples were completely removed (below 0.3 mg/L of nitrogen concentration) within 3 - 5 days. Ammonia concentrations in the AFBR effluent started to decrease from 30% recovery rate on day 1 to near 0% on day 10. Minimal concentrations of ammonium ions were detected throughout the subsequent experimental days (Figure 5.3). We were not able to establish complete nitrogen balance in the AFBR because N2 gas and

N2O, the well-known product and intermediates of microbial denitrification, were not measured in this study. In previous batch denitrification tests with mixed cultures and ZVI (Chapter 2), about 25 - 30% of removed nitrate-nitrogen was recovered as ammonia-nitrogen. In comparison, the AFBR significantly lowered ammonia concentrations in the treated effluent, thus enhancing the potential of integrated microbial-ZVI technology in environmental applications. We hypothesized that the enhanced nitrate reduction may be due to the establishment of anammox bacteria during the operation under the given condition with ammonium and nitrite remaining in the system by abiotic and biotic nitrate reduction. It has been reported that anammox cultures exists as red granules, which are distinguishable with the naked eye (Park et al., 2017). After 30 days of operation, the red color cultures was observed as a visual (but not conclusive) indicator the presence of anammox bacteria in the AFBR.

5.3.2.1 Characterization of Biofilms on Zero-valent Iron First, confocal scanning laser microscopy (CSLM) was used to evaluate the physical proximity between microorganisms and ZVI particles in the AFBR. Confocal imaging techniques are useful to examine microbial colonization and biofilm formation of ZVI surfaces because both microorganisms and iron surface can be simultaneously obtained (Jawerth et al., 2010). With confocal reflection microscopic (CRM) technique, the morphology of specimens were acquired (from the back- scattered light), whereas fluorescence signals use emitted light from the fluorophores in the sample. Figure 5.4 presents the digital confocal images of microbial colonies on iron surface acquired using confocal microscopy. Green fluorescence signals are individual

cells or small clumps of cells stained with SYTO 13 dye, and grey area was reflected light of ZVI surface. Confocal images show that the green dots or clusters, which were 1-2 μm in size, mostly associated with the iron surface (Figure 5.4(a)). At a higher magnification, the fluorescent-labeled microorganisms are located on the reflected surfaces, while none of fluorescence signals were detected in the solution phase (Figure 5.4(b)). The confocal images showed that microbial colonies were formed not only at the ZVI surface, but also within the crevices of ZVI particles. Scanning electron microscope (SEM) images of the ZVI surface were obtained to obtain high resolution visual images of biofilm. Biomass samples were pretreated according to an opportune dehydration method that resulted in a successful fixation and clear visualization of the bacterial cells. Electron micrograph of the ZVI sample from the AFBR shows that the ZVI surface was completely covered by the deposition of microorganisms and minerals (Figure 5.5). In contrast, fresh iron surface was clean and smooth (Figure 5.6). Bacterial flora on the ZVI surface consisted of rod-shaped and spherical bacteria. Most of bacteria appears to be tightly embedded in the biofilm on the ZVI surface.

5.3.2.2 Diversity of Microbial Communities in the Biofilm Concentrations of genomic DNA (gDNA) extracted from the centrifuged pellet and the ZVI surface were compared to estimate the degree of microbial colonization on the ZVI surface. Genomic DNA (gDNA) concentration extracted from the ZVI surface was almost 10 times higher than that from the pellet (Figure 5.7). This result indicates that the majority of the biomass in the AFBR are growing on the ZVI surface rather than in solution.

The purity of the DNA samples was also important for sequencing and it is commonly evaluated from the ratio of absorbance at 260 and 280 nm. If the ratio is around 1.8, the sample is generally considered as pure DNA. The A260/A280 ratio of gDNA sample extracted from iron particle was 1.86, while the ratio for the pellet gDNA was 1.41, indicating more contaminants are present. Therefore, only the ZVI extracted gDNA was used for 16S rRNA gene sequencing on the Illumina MiSeq platform. The gDNA extracted from seed cultures were also sequenced as the control. The hypervariable V4 region was amplified with the primer pair 515F/806R. The read length was targeted as 250 bp for the primers, followed by read processing and quality filtering, including denoising and chimera checking. The amplicon sequencing run yielded a total of 25,000 and 43,300 reads for the samples from the seed cultures and the ZVI surface, respectively. The sequencing results from RTL Genomics (Lubbock, TX) were presented in Krona charts (Figure 5.8 and 5.9), which illustrates the distribution of bacteria in seed cultures and the ZVI surface. The six most abundant phyla for both of samples included Proteobacteria, Bacteroidetes, Actinobacteria, Acidobacteria, Planctomycetes, and Chloroflexi (Figure 5.10). Proteobacteria was the most abundant phyla, and accounted for 51 and 62 % of bacteria in both samples. On the other hand, the distribution of the major phyla were different between two samples. Seed cultures have greater taxonomic diversity than ZVI-surface cultures as shown in Figure 5.10. In ZVI-surface cultures, 93% of bacterial populations belongs to Proteobacteria and Bacteroidetes phyla (Figure 5.10), which indicates a significant shift in microbial composition during the 50-day operation of AFBR. It has been reported that most

denitrifiers belong to the Proteobacteria phylum and some denitrifiers belong to Bacteroidetes phylum (Zumft, 1997; Braker and Conrad, 2011). Within Proteobacteria phylum, β-Proteobacteria was the most dominant taxa at the class level in the seed cultures while α-Proteobacteria was the dominant class the AFBR cultures. At the level of order, the relative abundance of Rhizobiales increased sharply from 5% to 35% from the seed cultures to AFBR cultures (Figure 5.11). The members of Rhizobiales also occupied more than 5 % of the total reads of the seed culture. Rhizobiales was identified as the most dominant order in the mixed culture samples from a long-term denitrification study (Grieẞmeier et al., 2017). In addition, the relative abundance of Xanthomonadales increased from 1 to 7% during the 50-day operation of AFBR. Bacterial species of Xanthomonadales order have been reported as one of common denitrifying species in wastewater treatment plant, soil, and lab- scale fluidized bed reactor (Wanner et al., 1990; Finkmann et al., 2000; Gentile et al., 2006; Saddler and Bradbury, 2005). About 5% of total reads in the seed cultures from Elkton Wastewater treatment was Planctomycetes, which are commonly detected in activated sludge of various wastewater treatment plants (Chiellini et al., 2013). The presence of Planctomycetes bacteria in our samples, however, are significant because all known anammox bacteria are currently belong to Planctomycetes phylum. The relative abundance of Planctomycetes in the ZVI cultures was less than 1 % of total reads. Even though the relative abundance of Planctomycetes in the ZVI culture is small and it is not possible to infer the ecological relevance of these microbial component with our results, the detection of Planctomycetes combined with the chemical data supporting anammox-like process suggests that low ammonia accumulation in the AFBR may be due anammox activity.

Schematic of anammox and denitrifying biofilm structure on ZVI surface is proposed in Figure 5.12. Anammox bacteria are strictly anaerobes and autotrophs and thus they are most likely grow in the inner layer of the biofilm near the ZVI surface. In addition to anammox bacteria, the ZVI surface are colonized by the co-existing denitrifying bacteria that supply nitrite for anammox activity while utilizing cathodic hydrogen and ZVI corrosion products as the electron donors. However, an excess growth of the co-existing bacteria may negatively affect the activity of anammox bacteria. Further studies are needed to clarify the spatial distribution of anammox biofilm and functions of the coexisting bacteria in the anammox biofilm.

pump

Figure 5.1. A schematic diagram of anaerobic fluidized bioreactor (AFBR) for denitrification test.

45 Nitrate 40 35 Ammonia

30 25 20 15 10

Concentration (mg N/L) (mg Concentration 5 0 0 1 2 3 4 5 6 Days

Figure 5.2. Nitrogen profile of the anammox-ZVI batch reactor.

Nitrate 30 Ammonia

10 Concentration (mg/L as N) as (mg/L Concentration 5

0 0 10 20 30 40 50 Days

Figure 5.3. Nitrogen concentration profile of AFBR.

(a) (b)

5 µm 5 µm

Figure 5.4. Confocal scanning laser microscopy (CSLM) images of ZVI surface retrieved from AFBR after 50 days operation. (a) low magnification. (b) high magnification. Bar, 5 µM.

2 μm

Figure 5.5. Scanning electron microscopy (SEM) image of ZVI surface retrieved from AFBR after 50 days operation.

Figure 5.6. Scanning electron microscopy (SEM) image of fresh ZVI surface.

10 Concentration (ng/ul) Concentration 5

0 Pellet ZVI

Figure 5.7. The concentration of extracted genomic DNA from centrifuged pellet and ZVI surface.

Figure 5.8. Krona chart of the sequencing results of bacterial taxonomic levels of extracted gDNA from the seed culture.

Figure 5.9. Krona chart of the sequencing results of bacterial taxonomic levels of extracted gDNA from AFBR (after 50 days operation).

100%

90%

80%

70%

Others 60% Chloroflexi Acidobacteria 50% Actinobacteria Bacteroidetes 40% Proteobacteria Planctomycetes 30%

20%

10%

0% Seed culture Up-flow ZVI Bioreactor

Figure 5.10. Relative phyla abundance of sequenced gDNA samples from seed culture and AFBR.

25 Burkholderiales

20 Rhodocyclales

15 Rhizobiales

Percentage (%) Percentage 10 Xanthomonadales 5

0 Seed culture Up-flow ZVI bioreactor

Figure 5.11. Relative order abundance in phyla Proteobacteria of sequenced gDNA samples from seed culture and AFBR.

Figure 5.12. Schematic illustration of the surface of ZVI and microbial colonization for enhanced denitrification with anammox.

Chapter 6

CONCLUSIONS

6.1 Summary of Results

6.1.1 Denitrification Performances in the Mixed-culture System with Various Electron Donors and Zero-valent Iron Biological denitrification under the presence of zero-valent iron (ZVI) as a source of electron donors was explored. Hydrogen gas and ferrous iron, which are the major products of anaerobic iron corrosion, were fed to denitrifying bacteria separately as a sole electron donors. Microbial-mediated reactors which were fed with

2+ H2, Fe , and ZVI completely reduced nitrate, even though they showed different reduction rates depending on the type of electron donor. Ammonium ion which is the product of abiotic nitrate reduction did not appear within the reactors of hydrogen gas or ferrous-fed cultures, while 30% of reduced nitrate was recovered as ammonia in ZVI-supported culture system. From the microbial diversity analysis using the data of 16S rRNA gene sequencing, it was identified that Dechloromonas sp., which is known to perform hydrogenophilic denitrification, was predominant species in both ZVI- supported and H2-fed cultures. Anaerobic nitrate respiration coupled to cathodic hydrogen oxidation was the dominant reaction in microbial-mediated ZVI-supported nitrate reduction process.

6.1.2 Enhanced Nitrate Removal by Microorganisms and Zero-valent Iron under Low Temperature Nitrate reduction tests at temperatures below 25 ℃ were performed under the presence and absence of microorganisms. ZVI-cell reactors showed complete reduction of nitrate around the ambient temperature above 10 ℃. At cold temperature (3.5 ℃), denitrifying bacteria still performed faster nitrate reduction with ZVI support than the abiotic reduction at the room temperature. The degree of nitrate reduction correlated with the concentration of soluble iron including ferrous and ferric iron. This iron concentrations were higher at the reactors with microorganisms than the reactors without them, even more at lower temperature. This confirmed that biological denitrification by bacteria accelerated the ZVI corrosion by consuming H2 gas film formed around ZVI for microbial metabolism, as in microbially induced corrosion (MIC) process. This would result in faster corrosion of iron in ZVI system with denitrifying bacteria than the reactor with only ZVI for nitrate removal.

6.1.3 Enhanced Nitrate Reduction by Anammox Bacteria in Iron-supported System A challenge of ZVI application to denitrifying system is requiring an additional treatment for ammonia. The application of anammox bacteria into the denitrifying- ZVI system resulted in the enhancement of denitrification in the ZVI-cell reactor tests with much less conversion of ammonia which was more favorable subsequently. Generally, the previous ZVI-denitrifying tests showed about 30% of ammonium recovery from the reduced amount of nitrate-nitrogen.

6.1.4 Microbial colonization on the Surface of Zero-valent Iron An anaerobic ZVI fluidized reactor was operated to understand the physical proximity of microbial-metal interface during the denitrification. This system showed

enhanced nitrate reduction compared to the other previous short-term batch tests. Based on the confocal and scanning electron microscopic images, it was identified that the microorganisms colonized the surface of iron particles as well as this changed the morphology of the surface. Also, the extracted genomic DNA concentration of ZVI surface was 10 times higher than the sample concentration of pellets from the mixed liquor. Therefore, microbial biofilm construction on the ZVI surface was essential not only for microbial growth, but also for enhanced nitrate reduction. ZVI was confirmed as a beneficial niche for microbial growth.

6.2 Recommendations for Future Work In this study, the efforts to understand the mechanisms and interaction between microorganisms and zero-valent iron during denitrification processes had been made. It was clearly confirmed that denitrifying bacteria utilizing various electron donors from anaerobic ZVI corrosion achieved efficient nitrate reduction under various circumstances (e.g., electron donors, low temperature, etc.). Also, the presence of anammox bacteria enhanced overall inorganic nitrogen removal by converting part of them to innocuous nitrogen gas from the water. The reduction kinetics and denitrifying performing bacteria were elucidated by well-performed chemical and microbiological characterization. It was meaningful to find the correlation between the anaerobic nitrate respiration and the enrichment of specific microbial community in the hydrogen-fed and ZVI-fed cells, even though denitrifying bacteria is phylogenetically heterogeneous microorganisms. During the denitrification tests, we were unable to measure nitrogen gas easily due to the difficulties in sampling and analysis. If it is necessary, an additional microbiological analysis, e.g., identification of functional gene (nar, nir, nor, and nos),

for every denitrification steps and their expression, would be followed to confirm enzymatic level of denitrification which can be another way to ensure microbial performances. In addition, to identify the actively denitrifying microorganisms in the ZVI biofilm, the similar microbiological techniques or tools would be used, too. Further studies can clarify to comprehend the spatial distribution of anammox biofilm and functions of the coexisting bacteria in the anammox biofilm. The performance of biological denitrification by the mixed culture were evaluated at lab-scale in this study. The pilot-scale studies is recommended to develop models for field applications. ZVI and its by-products from ZVI-denitrification are relatively less harmful, but it would be also suggested to accomplish an investigation on the environmental impacts of this technology.

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