Effects of ph and temperature on simultaneous nitritation, anammox and denitrification in a wetland treatment system receiving dairy wastewater

EFFECTS OF PH AND TEMPERATURE ON SIMULTANEOUS NITRITATION, ANAMMOX AND DENITRIFICATION IN A WETLAND TREATMENT SYSTEM RECEIVING DAIRY WASTEWATER by Yuling He A thesis submitted in partial

EFFECTS OF PH AND TEMPERATURE ON SIMULTANEOUS NITRITATION, ANAMMOX AND DENITRIFICATION

IN A WETLAND TREATMENT SYSTEM RECEIVING DAIRY WASTEWATER

by Yuling He

A thesis submitted in partial fulfillment

of the requirements for the Master of Science Degree State University of New York College of Environmental Science and Forestry

Syracuse, New York April 2011

Approved: Department of Environmental Resources Engineering

Wendong Tao, Major Professor Charles Maynard, Chair

Examining Committee

Charles N Kroll, Department Chair S Scott Shannon, Dean

The Graduate School

Gary Scott, Director, Division of

Environmental Resources Engineering

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my research will not be possible

I would like to thank all those who have contributed to the completion of this degree: Chris

Norton for helping on wetlands operation, Maria B Hosmer-Briggs for consulting on academic

writing and communication, and the examining committee members for their understanding and valuable comments

Finally, I want to thank my parents and sister for their continuous support, my boyfriend for his faithful encouragements and also suggestions on statistical analysis, my lovely friends for their smiles and words which helped me through many tough times

Table of Contents

List of Tables v

List of Figures vi

Abstract vii

Chapter 1: Introduction 1

References 3

Chapter 2: Literature Review 5

1 Nitratation Inhibition 5

2 Methods for pH Adjustment 9

3 Simultaneous Nitritation, Anammox and Denitrification 10

4 Fluorescent in situ Hybridization 11

References 12

Chaper 3: Manuscript 15

Abstract 15

1 Introduction 16

2 Materials and Methods 18

2.1 Wetland Treatment System 18

2.2 Field Measurements and Chemical Analysis 19

2.3 Fluorescent in situ Hybridization 19

2.4 Data Analysis 20

iv

3 Results and Discussion 21

3.1 Ammonium and Total Inorganic Nitrogen Removal 22

3.2 Effects of Temperature 23

3.3 Effects of pH 24

4 Conclusions 24

References 26

Tables 29

Figures 31

Appendix 35

Chapter 4: Conclusions and Recommendations 37

References 38

Vita 39

List of Tables

Table 1 Nitrogen removal rate of biofilters (BM1 and BM2) and free water surface wetlands

(CW1 and CW2) over four operational phases 29Table 2 Plant growth in free water surface wetlands 29Table 3 Relative abundance of AOB and anammox bacteria in biofilters and free water surface

wetlands at the end of Phase III 29 Table 4 Nitrogen removal performance in different wetland treatment systems 30

with higher pH values 34

Abstract

Y He Effects of pH and Temperature on Simultaneous Nitritation, Anammox and Denitrification

in a Wetland Treatment System Receiving Dairy Wastewater, 46 pages, 4 tables, 4 figures, 2011

A wetland treatment system, composed of two biofilters followed by two free water surface wetlands, was designed to enhance simultaneous nitritation, anammox and denitrification It was operated to evaluate effects of pH and temperature on nitrogen removal from dairy wastewater Furnace slag was utilized to increase pH Nitritation and anammox bacteria accounted for about 70% of the bacteria in each unit Temperature was the primary factor affecting nitrogen removal Significant pH effects were identified when temperature was below 18 ºC Ammonium removal rates were 1.10 and 0.86 g N/m2/d in the biofilters with pH at 8.2 and 7.7, respectively At raised

pH values (8.2-8.5) and temperatures (28.7 ºC on average), a biofilter removed 2.49 g N/m2/d of ammonium The free water surface wetlands removed ammonium at 3.10 g N/m2/d when

temperature was between 26.0 ºC and 13.8 ºC, and 1.24 g N/m2/d at temperature between 19.1 ºC and 15.1 ºC

Keywords: anaerobically digested dairy manure, anammox, biofilter, constructed wetlands,

fluorescent in situ hybridization, nitrogen removal, nitritation, denitrification

Y He

Candidate for the degree of Master of Science, April 2011

Wendong Tao, Ph.D

Department of Environmental Resources Engineering, Division of Engineering

State University of New York College of Environmental Science and Forestry,

Syracuse, New York

Wendong Tao, Ph.D. _

functional groups of bacteria Ammonium oxidizing bacteria (AOB) oxidize ammonia to nitrite (nitritation), while nitrite oxidizing bacteria (NOB) convert nitrite to nitrate (nitrataion):

Nitritation: NH4++ 1.5 O2→ NO2-+ H2O + 2H+ (1)

Both AOB and NOB are aerobic, consuming 4.25 g of O2 per gram of ammonia-nitrogen oxidized to nitrate–nitrogen Due to this high oxygen demand for ammonia oxidation, aeration is the main cost during this step (Ruiz et al., 2006)

In the denitrification stage, nitrate is reduced anaerobically to dinitrogen gas as equation 3 demonstrates:

8NO3- + 5CH3OH →4N2 + 10CO2 + 6H2O + 8OH- (3) Denitrifying bacteria are heterotrophic Sufficient soluble organic matter (electron donor) is needed to drive denitrification This increases the operating costs in wastewater treatment plants due to the cost of the additional carbon source and also the treatment of the surplus sludge that is generated (Gong et al., 2008)

In recent years, more attention has been paid to emerging and cost-effective biological

nitrogen removal processes such as nitritation coupled with anaerobic ammonium oxidation (anammox) (Strous et al., 1998):

Anammox: NO2- + NH4+ + 0.066HCO3- + 0.13H+ → 1.02N2 + 0.26NO3- + 2.03H2O +

0.066CH2O0.5N0.15 (4)

In anammox, nitrite formed by nitritation (Eq 1) acts as the electron acceptor to

anaerobically oxidize ammonium Since anammox bacteria are anaerobic the only oxygen

demand is from AOB Theoretically, nitritation - anammox consumes 63% less oxygen as needed for nitrification-denitrification process On the other hand, both AOB and anammox bacteria are chemoautotrophic that the oxidation of inorganic material does not yield as much energy as the oxidation of organic carbon sources by heterotrophic bacteria AOB and anammox bacteria, hence, have a slow growth rate and low cellular yield (Strous et al., 1999) Due to this characteristic,

nitritation-anammox process has no need for additional organic substrates and also produces less waste sludge

Dairy Wastewater and Constructed Wetlands

Over the years, with the growing size of herds, dairy farmers are faced with treatment for large amounts of manure including odor, nutrients, and pathogens Anaerobic digestion has been widely used in manure treatment, which converts organic carbon to biogas Although anaerobic digestion addresses both pathogen and odor issues and biogas can be used to generate heat and electricity, it has very poor ammonia-nitrogen removal (Uludag-Demirer et al., 2008) Therefore, the drainage from the agricultural land applied with digested manure contains high strength of nitrogen which may threaten the sustainability of soil, groundwater as well as surface water (Uludag-Demirer et al., 2005)

Constructed wetlands have provided a low-cost and low-maintenance alternative for treating high strength agricultural discharges (Knight et al., 2000) This type of engineered system is designed to utilize the natural processes involving wetland vegetation, soils, and their associated microbial assemblages to assist in treating wastewater They are designed to take advantage of many processes that occur in natural wetlands, but do so within a more controlled environment (Vymazal, 2007) Compared to conventional treatment methods, constructed wetlands are widely accepted for their economic and environmental benefits such as relatively low capital cost, low maintenance and no secondary pollution They can also provide food and habitat for wildlife while creating pleasant landscapes at the same time Constructed wetlands have two basic types:

subsurface-flow and surface-flow wetlands In subsurface-flow wetlands, water moves through a gravel or sand medium on which plants are rooted Unplanted subsurface-flow wetlands are similar to biofilters In surface-flow wetlands, water moves above the soil in a planted marsh or swamp Soil types include sand, silt and clay

Anammox has already been positively identified in constructed wetland treatment systems (Shipin et al., 2005) Obviously, the combination of constructed wetlands and anammox would be both economically and environmentally beneficial Research about anammox bacteria

performance in various types of constructed wetlands was carried out recently (Dong and Sun, 2007; Erler et al., 2008; Sun and Austin, 2007; Tao and Wang, 2009) However, nitrogen removal from anaerobically digested dairy manure (ADDM) in constructed wetlands enhancing anammox has not been reported

3

References

Chung, J., Shim, H., Park, S., 2006 Optimization of free ammonia concentration for nitrite accumulation in shortcut biological nitrogen removal process Bioprocess Biosyst Eng., 28, 275–282

Dong, Z., Sun, T., 2007 A potential new process for improving nitrogen removal in constructed wetlands—promoting coexistence of partial-nitrification and ANAMMOX Ecol Eng., 31, 69–78

Erler, D., Eyre, B D., Davision, L., 2008 The contribution of anammox and denitrification to sediment n2 production in a surface flow constructed wetland Environ Sci Technol., 42,

9144-9150

Gong, Z., Liu, S.T., Yang, F.L., Bao, H., Furukawa, K., 2008 Characterization of functional microbial community in a membrane-aerated biofilm reactor operated for completely autotrophic nitrogen removal Bioresour Technol., 99, 2749–2756

Knight, R.L., Payne, V.W.E, Borer R.E., Clarke, R.A., Pries, J.H., 2000 Constructed wetlands for livestock wastewater management Ecol Eng., 15, 41-55

Ruiz, G., Jeison, D., Rubilar, O., Ciudad, G., Chamy, R., 2006 Nitrification–denitrification via nitrite accumulation for nitrogen removal from wastewaters Bioresour Technol., 97, 330-335

Shipin, O., Kootatep, T., Khanh, N.T.T., Polprasert, C., 2005 Integrated natural treatment systems for developing communities: low-tech N-removal through the fluctuating microbial pathways Water Sci Technol., 51, 299–306

Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M., 1998 The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms Appl Microbiol Biotechnol., 50, 589–596

Strous, M., Kuenen, J.G., Jetten, M.S.M., 1999 Key physiology of anaerobic ammonium

oxidation Appl Microbiol Biotechnol., 65, 3248–3250

Sun, G., Austin, D., 2007 Completely autotrophic nitrogen-removal over nitrite in lab-scale constructed wetlands: Evidence from a mass balance study Chemosphere, 68, 1120-1128

Tao, W., Wang, J., 2009 Effects of vegetation, limestone and aeration on nitritation, anammox and denitrification in wetland treatment systems Ecol Eng., 35, 836-842

Tchobanoglous, G., Burton, F.L., Stensel, D.H., 2003 Wastewater Engineering: Treatment Disposal and Reuse, 4th Ed McGraw-Hill, New York

Uludag-Demirer, S., Demirer, G N., Chen, S., 2005 Ammonia removal from anaerobically digested dairy manure by struvite precipitation Process Biochem., 40(12), 3667-3674

Uludag-Demirer, S., Demirer, G N., Frear, C., Chen, S., 2008 Anaerobic digestion of dairy manure with enhanced ammonia removal J Environ Manage., 86(1), 193-220

Vymazal, J., 2007 Removal of nutrients in various types of constructed wetlands Sci Total Environ., 380(1-3), 48-65

5

Chapter 2: Literature Review

The following sections attempt to review possible methods to optimize and quantify

anammox performance in constructed wetland treatment systems

1 Nitratation Inhibition

To achieve a successful nitritation-anammox system, nitrite oxidizing bacteria (NOB) should stably be inhibited in the reactor to reserve nitrite for anammox process, while still maintaining the performance of ammonium oxidizing bacteria (AOB) at its maximum This is because NOB competes with AOB for dissolved oxygen also with anammox bacteria for nitrite and produces nitrate which is toxic to aquatic life A key factor for successful nitratation inhibition is to control nitrite oxidation suppressors Several studies have been carried out to investigate the effects of alkalinity, dissolved oxygen (DO), temperature, pH and free ammonia (FA, NH3) on nitratation inhibition

1.1 Dissolved Oxygen

The oxygen affinity constants of ammonia and nitrite oxidizers are 0.3 and 1.1 mg/L,

respectively (Wiesman, 1994), which provides a possibility of achieving nitratation inhibition by operating systems at low dissolved oxygen conditions Many studies were done to verify this hypothesis Ruiz et al (2003) proposed an optimum DO concentration of 0.7 mg/L The

experiment was carried in an activated sludge unit treating synthetic wastewater (610 mg NH4+N/L) Temperature and pH were maintained at 30 ºC and 7.85, respectively DO concentration was changed in steps from 5.5 to 0.5 mg/L by adjusting air flow level They found that below DO of 0.5 mg/L ammonium started to accumulate and over DO of 1.7 mg/L complete nitrification to nitrate occurred Similar results were also obtained in other research (Ciudad et al., 2005; Wu et al., 2008)

In aforementioned studies, the DO concentrations were accurately controlled by an air compressor or flow-meter, which is difficult for constructed wetland systems Although DO concentration could be adjusted to a favorable range by changing water depth or vegetation, DO concentration could only be used as an secondary factor in constructed wetlands

1.2 Alkalinity

AOB, NOB and anammox bacteria are closely interlinked with common electron donor and acceptors All the three types of bacteria are chemolithotrophic They require inorganic carbon for their cell growth By controlling alkalinity concentration, the elimination of NOB can be further enhanced In most of the applications involving anammox, alkalinity was adjusted by using chemicals like NH4HCO3, KHCO3 and NaHCO3 (Chung et al., 2006; Tang et al., 2010; van de Graaf et al., 1996) Stoichiometrically, nitrification consumes 7.14 g of alkalinity per g of NH4+-N

removed Bagchi et al (2010) proposed that an alkalinity consumption (alkalinity to ammonia

consumption) ratio of 7 or more is considered as an indicator of nitratation They found that maximum ammonia removal occurred at an influent alkalinity to ammonia ratio of 3.4 which corresponds with the theoretical alkalinity consumption of 3.6 mg/mg NH4+ N in the

nitritation-anammox process

1.3 Temperature

The ideal temperature for nitrification was from 20 ºC to 35 ºC (Hellinga et al., 1998) Dosta

et al (2008) reported maximum AOB activity at 35-40 ºC in short-term adjustment This strategy has been applied at the industrial scale in the SHARON process (Fux et al., 2002, Hellinga et al., 1998) However, there are also research pointed out that with proper adaptation, at temperatures higher than 20 ºC, it is possible for AOB to out-compete NOB because of their greater growth rate (Hao et al, 2002; Isaka et al., 2007) Nitratation inhibition could also occur when the temperature was as low as 18 ºC But if the temperature goes below 15 ºC, AOB would be affected as well

7

(Dosta, et al., 2008) For most of the non-tropical regions, temperature in constructed wetlands during most time over a year is expected to be below 30 ºC, which would not lead to nitratation inhibition

1.4 pH

Compared to DO concentration and temperature, pH control is more pragmatic in constructed wetland systems pH has a significant effect on nitratation inhibition, since AOB and NOB prefer different pH conditions In the low pH range NOB were predicted to grow faster than AOB

(Hellinga et al., 1998) Therefore, more researchers controlled pH to inhibit NOB by adding, for example, NaOH solution (Chung et al., 2006; Park et al 2007)

Park et al (2007) developed a bell-shaped empirical model to describe the pH-dependent behavior of maximum specific substrate utilization rates (MSSUR, ˆqpH) of ammonium and nitrite oxidation (Eq 1):

)(

)]}

(cos[

12

max

w pH pH w pH

pH pH w

q q

opt opt

opt pH

1.5 Free Ammonia (FA)

The proportion of ionized ammonium (NH4+) and free ammonia (NH3, FA) in liquid is affected

by pH and temperature FA concentration could be expressed by Eq.2 (Florida Department of Environmental Protection, 2001):

110

][

14

17]

N NH

FA , mg/L (2)

where pKa= negative logarithm of the acid dissociation constant for NH4+

Within the temperature range of 0oC-50oC and a pH range of 6.0 to 10.0, pKa = 0.0901821 + 2729.92/Tk, Tk = oC + 273.2

[NH 4 + -N] = Total ammonia concentration as N, mg N/L

It was reported that a certain concentration of FA at basic pH could restrain the activity of NOB However, AOB could be depressed at a high concentration of FA (Chung et al., 2006; Park, 2004; Ganigue et al., 2007) Therefore, the optimal range of FA concentration should not only stabilize nitratation inhibition but also maintain maximum ammonium removal

Chung et al (2006) investigated the optimal FA concentration range using sludge from two different sources, each with significantly different microbial distribution They estimated the conception of specific substrate utilization rates of ammonium oxidizers (qAO) and nitrite oxidizers (qNO) under various FA concentrations, with temperature, pH, and DO maintained at 30±0.5ºC, 8±0.5, and 5 mg/L, respectively

The highest AOB activity was observed when the FA concentration was 10 mg/L, and the rate decreased slightly with increasing FA concentration In case of NOB, the rate decreased

significantly with increasing FA concentration up to 5 mg/L NOB was more sensitive to FA than AOB They concluded that the optimal FA concentration range for both stable nitratation

inhibition and maximum ammonium removal appeared to be 5-10 mg/L for the adapted sludge This conclusion was in accordance with other studies (Abeling et al., 1992; Balmelle et al., 1992)

2 Methods for pH Adjustment

Ammonia oxidation is superior to nitrite oxidation at pH 8-8.5 (Park et al., 2007) Since H+ are continuously produced in nitritation process (Eq 1, Chapter 1), some slow-dissolving sources of alkalinity are needed to buffer pH

In the previous studies on anammox in constructed wetlands, pH was not controlled (Dong and Sun, 2007; Sun and Austin, 2007) The pH did not keep decreasing as H+ were produced by nitrification That could be attributed to photosynthesis of algae (Vymazal, 2007), the consumption

of H+ during anammox and the buffering of OH- produced by denitrification

Constructed wetlands are commonly used due to its relatively low capital cost, low

maintenance and no secondary pollution Therefore, chemical adjustments such as NaOH or HCl are not adaptable Tao and Wang (2009) applied limestone in surface-flow constructed wetland as

a natural source of alkalinity for anammox bacteria and pH buffer for nitritation Wetlands used limestone maintained significantly higher effluent pH (pH 8) than those used pavestone (pH 7.2) They concluded that although limestone in the rooting substrate made no significant difference from pavestone in ammonia and TN removal, it increased pH by 0.4-0.9, and raised the nitrite production peak from 3.6mg N/L to 4.7mg N/L, which created better conditions for anammox More long-lasting and cheap alkalinity sources that could be applied in wetlands for nitrogen removal were tested in 2008, by Renman et al They compared the pH adjusting ability of seven reactive filter materials i.e., coarse amorphous slag (ASC), crystalline slag, coarse (CSC) and very coarse (CSVC), limestone, opoka, Polonite® and sand Each material was filled to a height of 50cm in 30-cm wide columns The experiment was carried out indoors (temperature 20ºC) at the

Loudden wastewater treatment plant in Stockholm that receives domestic wastewater 0.5 L/h of wastewater was pumped from the plant and sprinkled equally over the column surface area every other hour for 67 weeks with a daily hydraulic loading rate of approximately 85 L∙m2/d All six materials except for sand maintained pH 8-9 stably

3 Simultaneous Nitritation, Anammox and Denitrification

Stoichiometrically, the anammox process removes only about 90% of the incoming

ammonium and produces nitrate which is about 10% of the influent ammonium Nitrate as a pollutant can affect human health (Kim-Shapiro et al., 2005) and can also be toxic to aquatic organism (Romano et al., 2007) Moreover, substantial experiments showed that oxygen and organic carbon can completely inhibit the anammox activity The inhibition from organic carbon is explained as follows: nitrite is an intermediary compound in nitrification and denitrification When nitritation produces nitrite, denitrification could start from this nitrite Once there is enough organic carbon present to support denitrifiers, anammox bacterial growth will be significantly suppressed due to the weaker competition for nitrite (electron acceptor) and living space (Tang et

al, 2010; Dong and Tollner, 2003) Thus, anammox process is usually successfully obtained under strictly anoxic and low organic carbon source conditions However, most of the ammonium-rich wastewater was produced with a certain concentration of COD Researchers then started trying to combine denitrification with nitritation -anammox processes for nitrogen removal from

wastewater with a high ammonium concentration and a low C/N ratio such as in municipal landfill leachate and poultry manure (Dong and Tollner, 2003; Wang et al., 2010) It was shown that anammox bacteria should not be threatened by denitrifiers when influent COD is as low as 100-150 mg/L (Tang et al., 2010; Chen et al., 2009) In this way, AOB consumes oxygen and creates anoxic environment for anammox and denitrification microorganisms Ammonium and COD with the small amount of nitrate produced by anammox (Eq 4, Chapter 1) are then removed through nitritation-anammox and denitrification, respectively This novel nitrogen removal

11

process is called SNAD (simultaneous nitritation, anammox and denitrification) The co-existence

of nitritation- anammox and denitrification were observed in constructed wetlands (e.g Dong and Sun, 2007; Tao and Wang, 2009) or other treatment systems (Chen et al., 2009; Wang et al., 2010) Several researchers also investigated the contributions of SNAD to TN removal (e.g Dong and Tollner, 2003; Pathak et al., 2007)

4 Fluorescent in situ Hybridization

Nitrogen removal efficiency of wastewater treatment systems is ultimately determined by the concentration and activity of related bacteria Therefore, to characterize the structure of

microorganisms in treatment facilities is necessary to help understand removal mechanisms and

optimize operation parameters Fluorescent in situ hybridization (FISH) is claimed to be the gold

standard for the detection of anammox organisms (Schmid et al., 2005) It is a cytogenetic

technique using fluorescent dye labeled rRNA oligonucleotide probes to detect and localize the presence or absence of specific DNA or RNA sequences on chromosomes Phylogenetic analysis

of anammox 16S rRNA sequences has shown that anammox bacteria form a monophyletic branch

within the phylum Planctomycetes Anammox organisms possess linked 16S and 23S rRNA genes,

which can serve as a target for FISH (Schmid et al., 2005) The bacteria that have been hybridized with the selected probes can be observed using fluorescence microscopy and ultimately

characterized in terms of the size and distribution of the bacteria, and the number of hybridized cells (Delatolla et al., 2009) It provides a cultivation-independent method of monitoring bacterial populations in biofilms

References

Abeling U., Seyfrid C., 1992 Anaerobic-aerobic treatment of high strength ammonium waste water-nitrogen removal via nitrite Water Sci Technol., 26, 1007-1015

Balmelle B., Nguyen K., Capdeville B., Cornier, J.C., Deguin, A., 1992 Study of factors

controlling nitrite build-up in biological processes for water nitrification Water Sci Technol., 26, 1017-1025

Bagchi, S., Biswas, R., Nandy, T., 2010 Alkalinity and dissolved oxygen as controlling parameters for ammonia removal through partial nitritation and ANAMMOX in a single-stage bioreactor J Ind Microbio, Biot., 37(8), 871-876

Chen, H., Liu, S., Yang, F., Xue, Y., Wang, T., 2009 The development of simultaneous partial nitrification, ANAMMOX and denitrification (SNAD) process in a single reactor for nitrogen removal Bioresour Technol., 100, 1548-1554

Chung, J., Shim, H., Park, S., 2006 Optimization of free ammonia concentration for nitrite accumulation in shortcut biological nitrogen removal process Bioprocess Biosyst Eng., 28, 275–282

Ciudad, G.,Rubilar, O., Munoz, P., Ruiz, G., Chamy, R., Vergara, D., Jeison, D., 2005 Partial nitrification of high ammonia concentration wastewater as a part of a shortcut biological nitrogen removal process Process Biochem., 40, 1715-1719

Delatolla, R., Tufenkji, N., Comeau, Y., Lamarre, D., Gadbois, A., Berk, D., 2009 In situ

characterization of nitrifying biofilm: Minimizing biomass loss and preserving perspective Water Res., 43, 1775-1787

Dong, Z., Sun, T., 2007 A potential new process for improving nitrogen removal in constructed wetlands—promoting coexistence of partial-nitrification and ANAMMOX Ecol Eng., 31, 69–78

Dong, X., Tollner, E W., 2003 Evaluation of anammox and denitrification during

anaerobic digestion of poultry manure Bioresour Technol., 86(2), 139-145

Dosta, J., Fernández, I., Vázquez-Padín, J R., Mosquera-Corral, A., Campos, J L., Mata-Álvarez, J., Méndez, R., 2008 Short- and long-term effects of temperature on the anammox process J Hazard Mater., 154(1-3), 688-693

13

Florida Department of Environmental Protection, 2001 Calculation of Un-Ionized Ammonia in Fresh Water Storet Parameter Code 00619 In: Chemistry Laboratory Methods Manual,

Tallahassee

Fux, C., Boehler, M., Huber, P., Brunner, I., Siegrist, H., 2002 Biological treatment of

ammonium-rich wastewater by partial nitrification and subsequent anaerobic ammonium

oxidation (anammox) in a pilot plant J Biotechnol., 99(3), 295-306

Ganigué, R., López, H., Balaguer, M D., Colprim, J., 2007 Partial ammonium oxidation to nitrite

of high ammonium content urban landfill leachates Water Res., 41(15), 3317-3326

Hao, X., Heijnen, J J., Van Loosdrecht, M C M., 2002 Model-based evaluation of temperature and inflow variations on a partial nitrification–ANAMMOX biofilm process Water Res., 36(19), 4839-4849

Hellinga, C., Schellen, A., Mulder, J., van Loosdrecht, M., Heijnen, J., 1998 The SHARON process: An innovative method for nitrogen removal from ammonium-rich waste water Water Sci Technol., 37(9), 135-142

Isaka, K., Sumino, T., Tsuneda, S., 2007 High nitrogen removal performance at moderately low temperature utilizing anaerobic ammonium oxidation reactions J Biosci Bioeng., 103(5),

486-490

Kim-Shapiro, D B., Gladwin, M T., Patel, R P., Hogg, N., 2005 The reaction between nitrite and hemoglobin: The role of nitrite in hemoglobin-mediated hypoxic vasodilation J Inorg Biochem., 99(1), 237-246

Park S., 2004 Multi-species nitrifying biofilm model including substrate inhibition and oxygen limitation PhD Dissertation, Hanyang University

Park, S., Bae W., Chung, J., Baek, S., 2007 Empirical model of the pH dependence of the

maximum specific nitrification rate Process Biochem., 42, 1671-1676

Pathak, B K., Kazama, F., Saiki, Y., Sumino, T., 2007 Presence and activity of anammox and

denitrification process in low ammonium-fed bioreactors Bioresour Technol., 98(11),

2201-2206

Renman, A., Hylander, L., Renman, G., 2008 Transformation and removal of nitrogen in reactive

bed filter materials designed for on-site wastewater treatment Ecol Eng., 34, 207-214

Romano, N., Zeng, C., 2007 Acute toxicity of sodium nitrate, potassium nitrate and potassium chloride and their effects on the hemolymph composition and gill structure of early juvenile blue

swimmer crabs (Portunus pelagicus, Linneaus 1758) (Decapoda, Brachyura, Portunidae) Environ

Toxicol Chem., 26, 1955–1962

Ruiz, G., Jeison, D., Chamy, R., 2003 Nitrification with high nitrite accumulation for the

treatment of wastewater with high ammonia concentration Water Res., 37, 1371-1377

Schmid, M.C., Maas, B., Dapena, A., van de Pas-Schoonen, K., van de Vossenberg, J., Kartal, B.,

et al., 2005 Biomarkers for In Situ Detection of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria Appl Environ Microbiol., 71(4), 1677-1684

Sun, G., Austin, D., 2007 Completely autotrophic nitrogen-removal over nitrite in lab-scale constructed wetlands: Evidence from a mass balance study Chemosphere, 68, 1120-1128

Tang, C., Zheng, P., Wang, C Mahmood, Q., 2010 Suppression of anaerobic ammonium

oxidizers under high organic content in high-rate Anammox UASB reactor Bioresour Technol

101, 1762–1768

Tao, W., Wang, J., 2009 Effects of vegetation, limestone and aeration on nitritation, anammox and denitrification in wetland treatment systems Ecol Eng., 35, 836-842

van de Graaf, A.A., De Bruijn, P., Robertson, L.A., Jetten, M.S.M., Kuenen, J.G., 1996

Autotrophic growth of anaerobic ammonium-oxidizing microorganisms in a fluidized bed reactor Microbiol., 142, 2187-2196

Vymazal, J., 2007 Removal of nutrients in various types of constructed wetlands Sci Total Environ., 380(1-3), 48-65

Wang, C., Lee, P., Kumar, M., Huang, Y., Sung, S., Lin, J., 2010 Simultaneous partial

nitrification, anaerobic ammonium oxidation and denitrification (SNAD) in a full-scale

landfill-leachate treatment plant J Hazard Mater., 175(1-3), 622-628

Wiesman, U., 1994 Biological nitrogen removal from wastewater Adv Biochem Eng

Biotechnol., 51,113-154

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accumulation in predenitrification biological nitrogen removal process Front Environ Sci Eng (China), 2(2), 236-240

temperature were examined for ammonia removal from anaerobically digested dairy manure Marble chips were packed in the biofilters to buffer pH and supplement alkalinity Furnace slag was applied to one biofilter to further increase pH to 8.2-8.5 to examine pH effects on nitratation inhibition and nitritation-anammox enhancement Furnace slag was also placed on rooting

substrates in the free water surface wetlands to buffer pH All four treatment units were drained

and fed every seven days Fluorescence in situ hybridization analysis confirmed the existence of

nitritation and anammox bacteria which accounted for about 70% of the microbial community in each unit Temperature, which varied between 14.3ºC and 27.9ºC, was the primary factor

affecting nitrogen removal pH effects were identified only when temperature was below 18 ºC in the biofilters Ammonium removal rates were 1.10 and 0.86 g N/m2/d in the biofilters with pH at 8.2 and 7.7, respectively Ammonium removal rates at the elevated pH values (8.2-8.5) were 2.49 and 0.7 g N/m2/d in the biofilters at raised temperatures (28.7 ºC on average) and room

temperatures (19.0 ºC on average), respectively The free water surface wetlands removed

ammonium at 3.10 g N/m2/d during phase II when temperature was between 26.0 ºC and 13.8 ºC, and 1.24 g N/m2/d during phase III and phase IV at temperatures between 19.1 ºC and 15.1 ºC

Keywords: anaerobically digested dairy manure, anammox, biofilter, constructed wetlands,

fluorescent in situ hybridization, nitrogen removal, nitritation, denitrification