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Agave fiber biofilter hybrid system on the denitrification-nitrification processes in wastewater treatment

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Title:
Agave fiber biofilter hybrid system on the denitrification-nitrification processes in wastewater treatment
Creator:
Serrano, Adriana ( author )
Place of Publication:
Denver, Colo.
Publisher:
University of Colorado Denver
Publication Date:
Language:
English
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1 electronic file (83 pages). : ;

Thesis/Dissertation Information

Degree:
Master's ( Master of Science)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Geography and Environmental Sciences, CU Denver
Degree Disciplines:
Environmental Sciences
Committee Chair:
Allen, Casey D.
Committee Members:
Bolhari, Azadeh
Barbour, Jon

Subjects

Subjects / Keywords:
Water -- Purification -- Nitrogen removal ( lcsh )
Urban runoff ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Review:
When wastewater is discharged in the water bodies without proper treatment, it can contribute to undesirable changes in the ecosystem as well as people. Nitrogen can be transformed into different components that might be toxic and promote the growing of aquatic plants. These components are removed by providing tertiary treatment. Biofilters (BFs) packed with organic material have demonstrated good removal of pollutants in the wastewater treatment. Thus, we evaluated the denitrification-nitrification processes n a hybrid systems of Bds with agave fiber as a filter media with municipal wasterwater from Durango, Mexico. Two laboratory-scale biofiltration reactors were used in two trails with four hydraulic loading rates (HLR=0.54, 0.80, 1.07, and 1.34 m3~2d~1), after three months of conditioning period with an HLR of 0.27 m3m~2d~1. In this step, all BFs were fed at the top with municipal wastewater. Then, the BFs were connected in series. The effluent of anaerobic Bfs (BF1 and BF2) fed the aerobic BFs (BF3 and BF4) performed in duplicate. They hybrid system showed a maximum removal efficiency of 96 percent in biochemical oxygen demand (BOD), 87 percent in chemical oxygen demand (COD). 80 percent in total suspended solids (TSS) and 4 logarithmic unites in fecal coliforms (FC). Low production of NO3~ was observed in the anaerobic step (7 mg L~1). High nitrification rates were observed in the aerobic BFs reaching concentrations of 222 mg L1. Finally, the BF hybrid system packed with agave fiber is suitable in the treatment of municipal wastewater because it removes pollutants that cause environmental problems and its effluents met the Mexican and U.S. standards. The effluents can be safely discharged in the environment and reused for irrigation, prior to disinfection. In order to obtain low nitrate concentration, it is suggested to probe the hybrid system with nitrification-denitrification processes.
Thesis:
Thesis (M.S.)--University of Colorado Denver. Environmental sciences
Bibliography:
Includes bibliographic references.
System Details:
System requirements: Adobe Reader.
General Note:
Department of Geography and Environmental Sciences
Statement of Responsibility:
by Adriana Serrano.

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University of Colorado Denver
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
902877169 ( OCLC )
ocn902877169

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Full Text
AGAVE FIBER BIOFILTER HYBRID SYSTEM ON THE DENITRIFICATION-
NITRIFICATION PROCESSES IN WASTEWATER TREATMENT
By
ADRIANA SERRANO
B.S., Technological Institute of Durango, 2010
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Master of Science
Environmental Sciences
2014


This thesis for Master of Science degree by
Adriana Serrano
has been approved for the
Environmental Sciences Program
by
Casey D. Allen, Chair
Azadeh Bolhari
Jon Barbour


The result of this thesis was derived from the project Optimization of
denitrification-nitrification processes with organic biofilters treating municipal
wastewater (Optimizacion de un proceso de nitrificacion-desnitrificacion en biofiltros
organicos tratando aguas residuales municipales, clave SIP20130563). It was realized
in the Environmental Laboratory of Centro Interdisciplinario de Investigacion para el
Desarrollo Integral Regional Unidad Durango of Instituto Politecnico Nacional
Unidad Durango, Mexico, directed by Professor Juan Manuel Vigueras Cortes.


Serrano, Adriana (Master, Environmental Sciences)
Agave Fiber Biofilter Hybrid System on the Denitrification-Nitrification Processes in
Wastewater Treatment
Thesis directed by Assistant Professor Casey D. Allen
ABSTRACT
When wastewater is discharged in the water bodies without proper treatment, it
can contribute to undesirable changes in the ecosystem as well as people. Nitrogen can
be transformed into different components that might be toxic and promote the growing
of aquatic plants. These components are removed by providing tertiary treatment.
Biofilters (BFs) packed with organic material have demonstrated good removal of
pollutants in the wastewater treatment. Thus, we evaluated the denitrification-
nitrification processes in a hybrid system of BFs with agave fiber as a filter media
with municipal wastewater from Durango, Mexico. Two laboratory-scale biofiltration
reactors were used in two trials with four hydraulic loading rates (HLR=0.54, 0.80,
1.07, and 1.34 m3m"2d_1), after three months of conditioning period with an HLR of
0.27 m3m"2d_1. In this step, all BFs were fed at the top with municipal wastewater.
Then, the BFs were connected in series. The effluent of anaerooic BFs (BF1 and BF2)
fed the aerobic BFs (BF3 and BF4) performed in duplicate. The hybnd system showed
a maximum removal efficiency of 96% in biochemical oxygen demand (BOD), 87%
in chemical oxygen demand (COD), 80% in total suspended solids (TSS) and 4
logarithmic units in fecal coliforms (FC). Low production of N03- was observed in the
anaerobic step (7 mg L'1). High nitrification rates were observed in the aerobic BFs
reaching concentrations of 222 mg L'1. Finally, the BF hybrid system packed with
agave fiber is suitable in the treatment of municipal wastewater because it removes
pollutants that cause environmental problems and its effluents met the Mexican and
IV


U.S. standards. The effluents can be safely discharged in the environment and reused
for irrigation, prior to disinfection. In order to obtain low nitrate concentration, it is
suggested to probe the hybrid system with nitrification-denitrification processes.
The form and content of this abstract are approved. I recommend its publication.
Approved: Casey Allen
v


DEDICATION
This thesis is dedicated to my parents who have dedicated their lives to my
education and well being and have supported me in every step I want to take. I also
dedicate this thesis to my loving grandparents that have always been supportive of me.
vi


ACKNOWLEDGMENT
I wish to thank my committee members that were more than helpful with their
expertise and precious time. A special thanks to Dr. Casey Allen for his patience
throughout the entire process and all the quality advising he was always provided.
Thank you to Dr. Azadeh Bolhari and Dr. Jon Barbour for advising in the different
aspects of the process and for agreeing to serve on my committee.
I would like to acknowledge the CIIDIR (Interdisciplinary Research Center for
Integrated Regional Development) in Durango, Mexico and specially Dr. Juan Manuel
Vigueras Cortes for providing all the resources and facilities for this projects
development.
Vll


TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION.........................................................1
1.1 Wastewater treatments...............................................1
1.2 Nitrogen cycle......................................................3
1.3 Identification of the problem.......................................6
1.4 General Objective...................................................9
1.6 Justification.......................................................9
II. LITERATURE REVIEW...................................................11
2.1 Biofilters (BFs)...................................................11
2.2 History of filters.................................................12
2.3 Advanced treatment.................................................14
2.4 Bio-filters packed with organic material...........................14
2.5 Importance of wastewater treatment.................................15
2.6 Characteristics of the municipality................................17
2.7 Principal activities...............................................18
2.8 Principal parameters of the wastewater.............................18
2.9 Established standards and guidelines...............................25
2.10 Previous studies..................................................27
III. METHODS.............................................................34
3.1 Hybrid system......................................................34
3.2 Filter material....................................................36
3.3 Wastewater influent................................................39
viii


3.4 Start-up period
40
3.5 Experimental procedure........................................40
3.6 Analytical methods............................................41
3.7 Experimental design...........................................43
IV. RESULTS AND ANALYSIS...........................................44
4.1 Wastewater characterization.................................44
4.2 Organic matter (BOD5 and COD) removal effect................44
4.3 Fecal coliform removal effect...............................50
4.4 Total suspended solids removal effect.......................53
4.5 Nitrates removal effect.....................................54
V. DISCUSSION.....................................................57
5.1 Organic matter (BOD5 and COD) removal effect................57
5.2 Fecal coliform removal effect...............................58
5.3 Total suspended solids (TSS) removal effect.................59
5.7 Nitrates removal effect.....................................60
VI. CONCLUSIONS AND RECOMMENDATIONS................................62
REFERENCES..........................................................64
IX


LIST OF FIGURES
Figure
1.Heterotrophic nitrification and aerobic denitrification.......................6
2 Seven Nitrogen Components.....................................................22
3 Schematic diagrams of the two combined anaerobic-aerobic BF systems..........34
4 Anaerobic-aerobic BF system...................................................35
5 Agave Duranguensis............................................................36
6 Agave fiber reception.........................................................37
7 Agave fiber before washing....................................................38
8 Preparation of the agave fiber................................................38
9 Biofilter single column.......................................................39
10 Wastewater collection........................................................40
11 Biofilters Columns...........................................................41
12 Effluents of the start-up period.............................................42
13 Effluents and influent of the last phase....................................42
14 Effluent BOD5 concentrations in the anaerobic BFs operated at four different
hydraulic loading rates..........................................................45
15 Effluent BOD5 concentrations in the aerobic BFs operated at four different
hydraulic loading rates..........................................................46
16 Air^HLR interaction on biochemical oxygen demand removal in the system at four
different hydraulic loading rates................................................48
17 COD average concentrations in the anaerobic biofilters......................47
18 COD average concentrations in the aerobic biofilters.........................47
19 Air effect in the Statistical analyses of COD removal in the hybrid system..49
20 HSL effect in COD removal in the hybrid system..............................50
21 Fecal Coliform concentrations in anaerobic biofilters........................51


22 Fecal Coliform concentrations in aerobic biofilters.............................51
23 Air effect in the Statistical analyses of FC removal in the hybrid system.......52
24 HLR effect of fecal coliform removal in the hybrid system.......................53
25 Total suspended solids concentrations in the two biofilter series...............54
26 Nitrate (N3_) concentrations in system 2 from BF2 to BF4.......................55
27 Air effect in the statistical analysis of N03_ removal in the hybrid system.....56
28 HLR effect of N03- removal in the hybrid system.................................56
xi


LIST OF TABLES
Table
4.1 Average composition of the raw wastewater...................................44
4.2 BOD average concentrations in the effluents..................................48
4.3 COD average concentrations in the effluents..................................50
4.4 Fecal coliform removal efficiencies in logarithmic units at four different hydraulic
loading rates....................................................................52
4.5 Total suspended solid effluent concentrations and removal efficiencies......53
4.6 N03_average concentrations in the effluents.................................55
Xll


CHAPTER I
INTRODUCTION
1.1 Wastewater treatments
Raw wastewater discharged from urban centers and municipalities represents
one of the main contributors to water pollution. Some of the consequences of polluted
water are changes in the growth rate of species, interferences with food chains,
increment of the toxicity levels, deterioration of peoples health, and impacts on
ecosystem services.
Water returned to the environment must satisfy many requirements. If
wastewater is discharged into the hydrosphere, it must be treated in order to decrease
toxic chemicals, pathogenic microorganisms, oxidizable compounds, and nutrients
that support microbial growth (Ganigue, et al.2007). Some examples of these
components that are most of concern are ammonia, nitrates, total coliform, and
infectious viruses.
The principal components of the municipal wastewater are derived from
domestic and commercial sources and include; human waste, solid and dissolved
forms of food waste, soaps and detergents, and soil residues. Municipal wastewater
treatment plants present fluctuations in the flow and contaminant concentrations
depending on human activities during the day (vanLoon and Duffy 2011). There are
different physical, chemical, and biological treatments that decrease contaminant
levels and allow the effluents to meet the regulations. The majority of these treatments
occur in municipal water treatment plants. Hybrid system occurs when two or more
processes are combined in series (Araujo, et al.2008).
Filtration by itself is an effective treatment that removes a large number of
contaminants. The innovative technology of biofiltration provides a proficient
1


treatment to the municipal wastewater. Thus, biofilters (BFs) can treat wastewater
from rural communities, agro-industry, schools, and even research centers. Organic
BFs do not require a large investment during their development, have a low operating
cost, are easy to operate, and take approximately one-fifth less space than a
conventional wastewater treatment plant (Garzon-Zuniga, Tomasini-Ortiz, et al.
2008). Lately, BF systems with organic materials have been used to remove organic
matter, suspended material, and pathogen organisms of municipal wastewater.
Activated carbon, one of the most popular materials, has excellent adsorption
efficiency but it is very expensive, increasing the cost of the wastewater treatment
(Riahi, Mammou and Thayer 2009). Additionally, other types of filter materials such
as peat and wood chips from different types of trees have been used in biofiltration
(Buelna and Belanger 1990; Lens, et al.1994; Garzon-Zuniga and Buelna 2011;
Gilbert, et al.2008). Natural fibers such as coconut fiber, date-palm fiber, bamboo
balls and agave fiber have also been used (Manoj and Vasudevan 2012; Riahi,
Mammou and Thayer 2009; Lens, et al.1994; Vigueras-Cortes, et al.2013). These
types of organic materials were selected based on their general characteristics,
availability, use, and cost. Recently, Vigueras-Cortes et al. (2013) obtained high
removal efficiencies in wastewater treatment with individual BFs using agave fiber as
a packing media.
Agave fiber can be obtained easily in the southeast of Durango State in
Mexico. A solid waste that is inexpensive, environmentally friendly, and available in
large amounts, the fiber waste is generated during mescal production (an alcoholic
beverage). During mescal production, the agave is cooked, compressed, and washed
to extract its sugars. In addition to its availability in this region, agave fiber has a wide
contact area that eases biofilm growth to remove wastewater pollutants. Vigueras-
2


Cortes et al.(2013) used agave fiber as filter material of a BF system where pollutant
removal was developed separately in aerobic and anaerobic conditions. The system
was performed using municipal wastewater from Durango City. It decreased
biochemical oxygen demand (BOD5), Total Suspended Solids (TSS), and helminthes
eggs (HE) efficiently with low cost, easy operation, and minimal space.
On the other hand, there are standards and guidelines for wastewater discharge
that have been established by the main environmental agencies in Mexico and the U.S
all based on physical, chemical, radiological, and microbiological properties as well
as in specific elements (vanLoon and Duffy 2011). Water quality guidelines depend
on the final use of the water. Usually, water used for human consumption requires
strict regulations whereas water reused for agricultural or industrial goals has less
strict regulations that do not account for removal of toxic chemicals and harmful
microorganisms.
1.2 Nitrogen cycle
Nitrogen, along with water, carbon, and phosphorus has its own cycle
(Withgott and Brennan, 2011). Nitrogen cycle has different processes such as,
nitrogen fixation, nitrification, anammox, and denitrification that transform nitrogen
into different forms. During nitrogen fixation, gas nitrogen is reduced to ammonia, a
biologically available nitrogen form. The biological reduction is catalyzed by
nitrogenase, a multimeric enzyme complex (Berman-Franka, Lundgren and Falkowski
2003). Thus, atmospheric nitrogen enters into the cycle mainly by nitrogen-fixation,
where it is converted to organic nitrogen (Latysheva, et al.2012).
Nitrification and denitrification are processes that occur naturally in the
environment, they occur in water, in soil, and in the atmosphere (M. S. Jetten 2008).
Because they remove undesirable nitrogen components, nitrification and
3


denitrification processes have been simulated in previous research (Rebah, et al.2010;
Li, et al.2013). Simulation of nitrification and denitrification processes can be
performed providing aerobic and anaerobic conditions. Thus, nitrogen components
that are dangerous to human health and the environment can be oxidized in simulated
scenarios.
In the nitrification process, ammonia is oxidized by the autotrophic nitrifying
bacteria in two steps. First, ammonia is oxidized to nitrite by ammonia-oxidizing
bacteria (AOB) in aerobic conditions and then nitrite is oxidized to nitrate by nitrite-
oxidizing bacteria (NOB) (Zhang, Love and Marc 2009). Nitrosomonas convert
ammonia under aerobic conditions to nitrite deriving energy from the oxidation
(Equation 1).Nitrite is oxidized by Nitrobacter to nitrate (Equation 2) (Posmanik,
Gross and Nejidat 2014):
2NH3 + 302 2N02~ + 2H+ + 2H20 (1)
2N02~ + 02 _vbaCter >2N3
The nitrate formed can be used positively as a nutrient for the plants.
Nevertheless, when there is an excess, it percolates into the water flowing through the
soil because it cannot retain the overabundance amounts of nitrate (Yoshimoto, et al.
2013). Thus, groundwater reservoirs can enclose nigh concentrations of nitrates which
cause extensive environmental problems. During this stage, nitrogen forms change but
are not assimilated. Nitrification is important for three main reasons:1)nitrification
represents a vital phase in the nitrogen cycle because of the production of nitrate, 2) if
nitrite is not oxidized, it can negatively affect peopled health, and 3) nitrifying
bacteria are usually competing with primary producers, so due to nitrification,
4


nitrifying bacteria are present only when ammonium is present in high concentrations
(Dodds 2002).
Denitrification is the other part of the nitrogen cycle. It develops the biological
reduction of nitrate to nitrogen gas (N2) by facultative bacteria. This process is
developed under anaerobic conditions where the bacteria use nitrate as the primary
source of oxygen (Busigny, et al.2013). The anoxic conditions for denitrification
require a dissolved oxygen concentration of maximum 0.5 mg/L. When the bacteria
break down nitrate in order to take the 02, nitrate is reduced to nitrous oxide (N2)
and then it turns to nitrogen gas (Equation 3) (Mateju, et al.1992)
N03 N02 NO + N20 N2 (3)
Nitrogen gas (N2) has low water solubility. Thus, it escapes to the atmosphere
as gas bubbles (Harter, et al.2014). Since N2 is the principal component of air, small
releases do not cause environmental concern.
During denitrification, a carbon source is required and in wastewater
treatments, the carbon source is obtained from the wastewater. Organic carbon is
oxidized to C02 and cellular energy (Equation 4), with water and hydroxyl ions as
end products (Mateju, et al. 1992)
6N03 + 5CH3OH ^ 3N2 + 5C02 + 7H20 + 60H (4)
Nitrification takes place under aerobic conditions, however, according to Wen
and Wei (2011)anaerobic nitrification of ammonia can be performed by heterotrophic
5


bacteria that use the organic matter (reductases) of the wastewater as the carbon
source.
Denitrification process under aerobic conditions was demonstrated by Wen
and Wei (2011), Shi, et al.(2013), Chen and Ni (2012), and Joo, Hirai and Shoda
(2005)also shown in figure 1.
AMO HAO NIR
nh3 ^ nh2oh ^ no2
HAO ^ NAP
N03
Reductases: Ammonia monooxygenase (AMO), hydroxylamine oxidase (HAO), periplasmic nitrate
reductases (NAP), nitrite reductases (NIR), nitric oxide reductase (NOR) and nitrous oxide reductase
(NOS) (Wen and Wei 2011).
Figure 1 Heterotrophic nitrification and aerobic denitrification
Nitrification and denitrification processes occur naturally in the environment
in such mediums as water, in soil, and in the atmosphere (M. S. Jetten 2008). Because
they remove undesirable nitrogen components, nitrification and denitrification
processes have been simulated in previous research (Rebah, et al.2010) (Li, et al.
2013). Simulation of nitrification and denitrification processes can be performed
providing aerobic and anaerobic conditions. Thus, nitrogen components that are
dangerous to human health and the environment can be oxidized in simulated
scenarios.
1.3 Identification of the problem
The development and improvement of methods for biological wastewater
treatment are very important, particularly methods that remove nitrogenous
contaminants. High concentrations of some nitrogen forms can cause significant
NOR
NOS
n2o
n2
6


atmospheric, terrestrial, and aquatic problems (Harter, et al.2014). The removal of
nitrogen components in wastewater represents a tertiary treatment but it is essential to
reduce their concentration in the effluents to discharge in receiving bodies.
During degradation of organic matter, an aerobic BF by itself produces
different nitrogen-nitrate (N-NO3") components. Ammonia-nitrogen and nitrates have
serious effects on the environment and human health. In human health, ammonia
irritates tissues and alters the uptake of oxygen by hemoglobin decreasing
oxygenation of tissues (Holeton, Chambers and Grace 2011). High levels of nitrate in
waters are also considered a threat to the public health because they can be ingested
by eating vegetables or drinking water. Drinking water containing large amounts of
nitrate causes methemoglobinemia in infants in 1962 (Vigil, et al.1965).
Methemoglobinemia is derived from the interaction between nitrite and hemoglobin
where nitrate converts to methaemoglobin-producing nitrite. This causes hypoxaemia
due to an oxygen dissociation curve (Chan 2011). High nitrates levels also increase
cancer risk due to reduction of nitrates to nitrites that can later form N-nitroso
compounds (NOCs). NOCs are dangerous carcinogens and act systematically (Njeze,
Dilibe and Ilo 2014).
In the environment, the presence of ammonium and nitrates-nitrogen in large
amounts in reservoirs, subsoil, and groundwater can cause catastrophic deterioration
of fresh water quality (Guerra, et al.2013). All natural bodies of water are able to
oxidize organic matter when the nitrogen loading -principally ammonia- is maintained
within the limits of the oxygen resources. When the nitrogen loading exceeds the
oxidative capacity of the water bodies, degradation of nitrogen forms is inhibited.
Acceptable levels of dissolved oxygen, calculated through research, also determine if
forms of aquatic organisms can survive (Rezaei, et al.2013).
7


In the past, oxygen sources were the pollutants with higher concern in surface
waters, but nowadays, pollution of surface and groundwater with contaminants from
industrial and agricultural sources is the highest concern. During the last 50 years, a
variety of chemical have been developed for agricultural purposes, such as fertilizers
and pesticides, that are principally composed of nitrogen and phosphorous
components (William, Borjesson and Hedlund 2013). Nitrogen forms are essential
fertilizers to the algae growth. Algae growth is not really environmentally desirable
and it can occur when water with nitrogen forms are discharged into bodies of water
(Li, et al.2013). If wastewater with ammonia and nitrate-nitrogen is discharged into
lakes, streams, or wetlands, it can cause eutrophication the process of an ecosystem
becoming more productive by nutrient enrichment stimulating primary producers
(Dodds 2002). Nutrients such as inorganic nitrogen promote the growth of undesirable
aquatic plants and algae. Algal blooms growth is one of the worst effects of
Eutrophication. They cause undesirable taste, odor, and color issues and increase
when lakes become more eutrophied (Martinez-Lopez, et al. 2007).
The oxidation of rivers and estuaries is another problem caused by dissolved
inorganic nitrogen forms. During the autotrophic conversion of ammonia to nitrate,
oxygen is required. Thus, when ammonia is discharged into rivers and estuaries, its
oxidation can cause a reduction of dissolved oxygen levels (Bresler 2012) which can
be even worse when long residence time for the growth of nitrifying bacteria is
available.
Ammonium nitrogen exists as un-ionized ammonia (NH3) or ionized ammonia
(NH4+). Un-ionized ammonia is more toxic than NH4+ because it is uncharged and
lipid soluble (Gao, et al.2011). In addition, toxicity is also determined by pH levels.
Concentrations above 0.2 mg/L of free ammonia can cause death in many species of
8


fish and the National Research Council Committee in the U.S. recommends this
amount as the limit permitted in receiving bodies (Sawyer, McCarty and Parkin
2003).
Thus, wastewater should be treated in order to decrease concentrations of
inorganic nitrogen forms that may damage the environment and human health.
1.4 General Objective
The main objective of this study rests in evaluating the denitrification and
nitrification processes in a hybrid system or biofilters using agave fiber as filter media
with four different superficial hydraulic loads with the intent of meeting Mexican and
US EPA effluent regulations.
This research intends to specifically improve the knowledge and application of
BFs by:
Determining the removal efficiencies of organic matter, suspended
material, nitrates and ammonia-nitrogen in the wastewater treatment
when anaerobic and aerobic conditions are provided and
Determining the maximum hydraulic loading when the effluents meet
the regulations of the municipal wastewater treatment and the effluents
can be reused in irrigation crops and green areas.
1.6 Justification
The organic BFs will contribute to decrease pollution and environmental
problems of receiving bodies by the discharge of treated effluents without nitrogen
components. Discharging effluents where nitrogen compounds have been removed
will decrease problems such as eutrophication, oxidation, and high levels of toxicity
in rivers, lakes, and estuaries (Cotman, Zagorc-Koncan and Drolc 2001). Hybrid
9


biofiltration systems are inexpensive and sustainable method for this process that can
be applied to decrease the impacts of wastewater discharges. The principal goal of
removing nitrogen compounds from wastewater is the protection of the environment.
In addition to the discharge of water without pollution, the water treated in this
system can be reused in productive activities such as industry and agriculture, in
places with lack of treatment plants and scarcity of water. Regulations and quality
guidelines are less strict for industrial purposes and irrigation. They are based on the
concentration of soluble salts, and potentially toxic elements as well as the molar ratio
of sodium to calcium and magnesium (USA-EPA 2012). All these chemical
components are in very low quantities or not contained in effluents. In order to prove
the low concentrations of these chemicals, conductivity and alkalinity analyses were
developed. Aesthetics criteria are generally unnecessary, however, odor and color are
very suitable in the wastewater treated by the organic BFs.
10


CHAPTER II
LITERATURE REVIEW
This section outlines specific and relevant literature related to this research
agenda. First, I defined BFs and their main category followed by the historical
development of filtration in wastewater treatment. Then, after discussing a vast
history of filters I briefly discuss advanced treatments and introduce the first BFs
packed with organic material. Subsequently, the importance of wastewater treatment
is described to continue with the characteristics and principal activities of Durango
municipality that provides a general idea of the possible wastewater constituents in
this area. The principal parameters in the wastewater are described as well as the
established standards and guidelines of the environmental agencies. Finally, previous
studies using organic BFs or/and aerobic and anaerobic conditions and achieving
nitrification and denitrification are presented.
2.1 Biofilters (BFs)
The trickling filter (TF) is a fixed biological bed of rock or plastic media on
which wastewater is applied for aerobic biological treatmentWang, et al.2009).
These attached growth systems use a media of granite, limestone, clinkers, plastic
tubs, or hard coal that distribute and contact the wastewater. A biological slime layer
called biofilm is grown in the filter media. The removal of the dissolved organic
pollutants takes place due to their biological oxidation developed by the slime film
and their followed degradation by the aerobic and facultative bacteria. The biological
filter (BF) is a type of trickling filter that is able to operate aerobically and
anaerobically. The biofilm contains microorganisms, particulate material, and
extracellular polymers. In addition to the inorganic media that trickling filters use,
organic media such as fibers, compost and wood are also used to pack biofilters.
11


Organic packing material is a good supporting material that allows the attachment of
microorganisms (Rebah, et al.2010). Degradation of organic matter and other
pollutants and retention of suspended solids have been successfully developed in
these systems.
2.2 History of filters
The trickling filter was installed for the first time at the Lawrence Experiment
Station in Massachusetts in 1891. It had a distribution by spray nozzles (Wang, et al.
2009). However, it was during the 20th century that the majority of improvements in
wastewater management were developed. In 1940biological filtration had a great
impact and most of the wastewater treatment plants in the United States had trickling
filtration. In 1946 the National Research Council developed a mathematic formulation
for the trickling filter design (Daiggerl and Boltz 2011). In addition, the membrane
bioreactor (MBR) which was one of the most important tools in the treatment of waste
liquids and solids was introduced in the mid-1960fs. Some of the advantages of MBR
are; high removal of pathogens and contaminants, minimal footprint, few energy
demand, low cost, and easy operation (EPA 2007). The MBR treatments include two
steps; bacterial degradation of organic matter in the presence of dissolved oxygen
developed in a bioreactor, and the separation of suspended solids and bacteria from
the effluent developed by the membrane in a second step (Sutherland 2010). These
processes make up the complete treatment -primary, secondary and even part of the
tertiary treatment-that is developed in a conventional wastewater treatment plant.
Dorr Oliver introduced the idea of combined sludge digestion into a very fine
filter with flat plate membranes in a side-stream loop in the mid-1960s. In this decade,
microfiltration and ultrafiltration membranes were developed at a commercial scale
(Baker 2000). However, the emerging technology was very expensive. It wasn5t until
12


1989 in Japan that the MBR started to be widely used due to the initiative of the
Japanese government to find efficient and low cost methods to wastewater treatment.
This new version emerged in the European market in the mid-1990s. After the 19905s
the MBR field experienced extensive growth. Many researchers started developing
new technologies in this field. Today there are more than 3000 plants around the
world that use this technology (Stephenson, et al.2000).
While MBR was expanding, the trickling filter experienced some
improvements. In the early 1960s, Imperial Chemical Industries, Ltd. initiated the
use of plastic media in TF. Plastic media allows the growth of the biofilm in its area.
Plastic materials were a success because of the high-rate trickling filter media that
was obtained (Wang, et al. 2009).
The anaerobic biofilter was another improvement to the attached growth
wastewater treatment. It was studied in 1962, for the first time by James Young and
Perry McCarty (Kaiser, Dague and Harris 1995). The anaerobic biofilter was packed
with different types of filter media. No oxygen was provided to this system, which
decreases energy use. During this biofiltration, an anaerobic carbonaceous oxidation
of the organic matter is performed. Anaerobic biofiltration was used to remove
nitrogen components because it provides suitable conditions to achieve denitrification.
However, improvements in the nitrification process are still needed. High
carbonaceous removal at low cost and low sludge production is achieved during
anaerobic biofiltration.
The biofilter performs the same process as MBR but it does it in one-step. The
packing material allows the growth of a biofilm and the suspension of organic matter.
Thus, the degradation of pollutants is developed by the attached microorganisms and
the suspended solids are trapped in the packing material.
13


2.3 Advanced treatment
Due to the understanding of the impact of wastewater and their pollutants such
as phosphorous and nitrogen compounds, advanced treatments to these pollutants
were developed. Once the reduction of carbonaceous and other pollutants was
achieved through the development of secondary treatment, the prevention of
eutrophication by the phosphorous and nitrogen compounds was the next step in the
history of wastewater treatment. The firsts in developing the denitrification process to
the wastewater treatment were Ludzack and Ettinger (1962). They used an anoxic
zone to achieve biological denitrification in an activated sludge process. Barnard
(1973) developed and patented a single sludge system that biologically removed
nitrogen and phosphorous (Lofrano and Brown 2010).
2.4 Bio-filters packed with organic material
In 1987, the use of organic media in biofiltration was studied. Rana and
Viraraghavan (1987) developed a biofilter packed with peat in The U.S. and Canada
that showed a good removal of pollutants, even nitrogen. During the second half of
the 1990fs and the beginning of the 2000s biofiltration through organic media was
widely performed. Organic BFs were applied in rural and semi-urban regions where
there were no wastewater treatment plants and the wastewater could be treated in situ.
The organic materials used in biofiltration were commonly endemic of the area where
the biofilters were developed. For example Garzon-Zuniga, Tomasini-Ortiz, et al.
2008 developed a trickling biofiltration in Morelos, Mexico using sugar cane fibers
which are very common in this area. Riahi, Mammou and Thayer 2009 developed a
BF in Tunisia packed with Phoenix dactylifera which stretches from North Africa to
the Middle East. There are many other examples of organic BF that are described
below.
14


2.5 Importance of wastewater treatment
Wastewater from industry and domestic use should meet a specific set of
requirements before being returned to the natural environment. The levels of toxic
chemicals and microorganisms contained in wastewater depend on the source because
the ecosystem is not able to transform high levels of pollutants. In some cases,
municipal wastewater that contains domestic and industrial wastes is not treated
before to discharge into water bodies such as rivers, lakes, and oceans. The typical
components of municipal wastewater are human waste, food waste, soaps, detergents,
and soil residues. The level of pollution in wastewater is determined by measuring
parameters such as biochemical oxygen demand (BOD5), chemical oxygen demand
(COD), total suspended solids (TSS), Total phosphorous (TP), and total nitrogen
(TN). Thus, when wastewater is discharged into the hydrosphere it should not contain
high levels of toxic chemicals dangerous to the ecosystem, oxidizable components,
nutrients that can cause microbial growth or pathogens (vanLoon and Duffy 2011).
Levels of BOD5, TSS, TP, and TN are the parameters with the greatest concern. These
parameters indicate the concentration of pollutants that affect the environment and
interfere with the marine life.
When wastewater is going to be used for irrigation, it is not required to remove
nutrients or benign dissolved and suspended organic matter. However, toxic
chemicals and microorganisms represent undesirable components and should be
removed (U.S. Environmental Protection Agency 2012). When wastewater will be
discharged in to the hydrosphere, in addition to toxic chemicals and microorganisms,
nutrients and dissolved organic matter need to be removed. Water bodies have
sufficient levels of dissolved oxygen to oxidize animal and vegetative wastes through
aerobic microbial reactions. During this process, dissolved oxygen is converted to
15


C02 (vanLoon and Duffy 2011). Carbon dioxide is then used during the
photosynthesis and converted again to oxygen. This natural cycle keeps the natural
ecosystems in a stable state. When excessive amounts of dissolved oxygen are
provided by wastewater discharge, the self-purification cycle is broken (Sonune and
Ghate 2004). Excessive amounts of degradable organic matter produce anoxic
conditions which reduces important aerobic biological degradations. The TSS of the
untreated wastewater cause an increase of turbidity which inhibits photosynthesis.
Nutrients such as phosphorous, nitrogen, and silicate are important elements to
the aquatic systems. Most of the plants use them for growth and reproduction (Biszel
and Uslu 2000). However, the enrichment of nitrogen and phosphorous has negative
effects on aquatic ecosystems. High levels of nitrogen and phosphorous stimulate
plant and algal growth. Inorganic nitrogen and phosphorous become available for
phytoplankton production in aquatic systems heavily contributing to eutrophication.
Nitrogen can be present in wastewater discharges through different inorganic nitrogen
forms such as ammonia nitrogen, nitrates, and nitrites. As it was mentioned before,
dissolved inorganic nitrogen forms can cause oxidation of rivers and death in many
species of fish.
In order to protect aquifers, regulations have been established by the
government where permissible levels of contaminants are stated. Considering the final
use, the effluents of treatment facilities are required to meet specific standards. The
established standards and guidelines are described later. Because of these guidelines,
a variety of wastewater treatments processes have been developed in order to clean
water and achieve effluent regulations. The typical treatment includes a primary and
secondary treatment where suspended particles are removed and organic matter is
converted to bacterial biomass, H20 and C02. Some processes include a tertiary
16


treatment where disinfection is performed or specific contaminants such as nitrogen
and phosphorous are removed (vanLoon and Duffy 2011). These treatments include
activated sludge process, chemical coagulants for turbidity removal, sludge digestion,
and attached growth systems. They may include physical, chemical, and biological
processes. Some of them have been established in big scales treating up to 10,000 m3
of municipal wastewater every day (Tchobanoglous, Burton and Stensel 2004). All
these processes and designs are frequently improved or replaced.
2.6 Characteristics of the municipality
The municipality of Durango is located in the Valle del Guadiana in the north
of Mexico. The word Guadiana means Wide River in the Arabic etymology. Due to
The Tunal River located in the Valle del Guadiana, this name was assigned to the area
since the first human settlement during the Colonial era. Durango City is the most
important city in the municipality of Durango and is also the capital of the state that
bares the same name. Durango is located at 24.2 north latitude and 104.4 west
longitude with an elevation of 1,880 m above sea level. The climate in Durango is
semi-arid with low precipitation during the summer and semi-cold during the winter.
The average temperature is 17.5C (INEGI 2013).
Durangos geology comprises the Cenozoic (70.9%)the Quaternary (23.6%)
the Neogene (4.1%), and the Paleogene (0.1%) periods. Recent alluvial deposits,
basaltic rocks, and extrusive igneous rocks can be found in the area (INEGI 2013).
Durango is the city with the biggest population in the state of Durango. At the end of
the eighteenth century, -three centuries after the official foundation of Durango- the
city had a population of 7400 inhabitants in 1500 houses. The streets where traversed
by canals that carried water from the wells to domestic use and irrigation. During the
1900s water was provided to the population through pressure pipes. It wasnt until
17


1969 when the potable water and sewage systems were introduced to some places of
Durango City. In 1969 the potable water and sewage systems had 57,370 m and
95,915 m of pipe network respectively. In 1987 the pipe network of sewage was
extended to 175,494 linear meters and 3,400 hookups where added to the 12,500 that
previously existed. During this period, 6 oxidation ponds to wastewater treatment
were built on an 86 hectares plot of land (Aguas del Municipio de Durango 2013).
The oxidation ponds were located where the municipal wastewater treatment plant is
currently situated. The wastewater used in this project was collected from the
municipal wastewater treatment plant in Durango City.
2.7 Principal activities
The sewage system indirectly provides information of the characteristics -
customs and behaviors- of the population from food to hygiene habits as well as from
the use of pharmaceuticals and birth control pills (Lofrano and Brown 2010). The
majority of the municipal wastewater from Durango comes from domestic and
commercial use. Industrial use is also included in the sources of municipal wastewater
but it does that in few quantities.
VanLoon and Duffy 2011 affirm that the highest level of flow and
contaminant concentration in the sewage are found during the morning and evening.
In the specific case of Durango, Mexico, due to the populations activities, higher
values of contaminant concentration of the municipal wastewater treatment plant are
found at 2:00 pm and 6:00 pm (Vigueras-Cortes, et al.2013).
2.8 Principal parameters of the wastewater
When developing wastewater treatments reducing the occurrence of specific
parameters remains a key goal. These parameters include: biochemical oxygen
demand, chemical oxygen demand, total solids, total nitrogen, and fecal coliform.
18


2.8.1 Biochemical Oxygen Demand (BODs)
BOD is the amount of dissolved oxygen required by bacteria to degrade
decomposable organic matter in aerobic conditions (Sawyer, McCarty and Parkin
2003). This parameter indicates the organic quality of the water by the determination
of biodegradable organic compounds (Riedel,et al.1988). The BOD test indicates the
oxygen that is required by the aquatic ecosystem to oxidize the organic matter once it
is discharged. If the BOD level is high, it inhibits the purification capacity of the
water systems. Thus, BOD data should be interpreted in terms of organic matter and
as the amount of oxygen used during its oxidation. The oxidation reactions are
governed by the number of microorganisms as well as the temperature. The BOD test
is performed in a constant temperature of 20C that is a usual value in natural bodies
of water. The complete organic matter oxidation theoretically takes an infinite amount
of time. However, for practical purposes, 20 days may be enough for complete
oxidation. Although 20 days sounds more practical, it remains a substantial period of
time. After many experiments, it has been found that in 5 days a large percentage of
the total BOD is exerted. Thus, a BOD test with a 5-day incubation period is the
current basis for determination of this parameter (Sawyer, McCarty and Parkin 2003).
This method is called the BOD5. It is important to keep in mind that only a portion (70
to 80 percent) of BOD is determined by this method due to the length of the period of
time that it is performed in. Another advantage of the BOD5 is that during the 5-day
incubation period it minimizes the possibility of oxidation of ammonia. The typical
BOD5 in municipal wastewaters is between 100-300 mg L 1 and depends on previous
water use (vanLoon and Duffy 2011). The goal of BOD in a wastewater treatment is
below 30mg L 1 which is the permissible level of regulations (The Clean Water Act
and NOM-003-SEMARNAT-1997).
19


2.8.2 Chemical Oxygen Demand (COD)
The COD is measured in order to determine the organic strength of domestic
and industrial wastes. It represents the total quantity of oxygen required for oxidation
of organic matter in order to obtain carbon dioxide and water. This oxidation is
known to be performed in all organic compounds by the action of strong oxidizing
agents under acid conditions. During COD determination, all the organic substances
are oxidized even if they are not able to assimilate biologically. The amount of
oxygen used during the oxidation process is related to the amount of organic matter
contained in the wastewater. Thus, COD values are higher than BOD values. When
wastewater contains high amounts of biologically resistant organic matter, COD
values are greater because of the amount of oxygen required for the oxidation to be
high. Glucose and lignin are examples of organic substances that require large
amounts of oxygen to oxidize.
Measuring BOD provides more advantages than measuring COD. The COD
test is less suitable because of the limitation differentiating biologically oxidizable
and inert organic matter. COD results provide an estimate of the amount of all organic
matter types together. COD test does not provide a real rate of organic matter
oxidation as it occurs in nature. However, COD requires less time (3 hours) to
measure than BOD. COD values can be interpreted in terms of BOD values by
establishing a reliable correlation between COD and BOD. The typical COD in
municipal wastewaters is 500 mg L"1 and it depends on previous water use (vanLoon
and Duffy 2011).
2.8.3 Total Solids
Solid matter is all of the matter contained in a liquid material except for the
water. However, solids are defined as the matter that remains as residue upon
20


evaporation and drying at 103-105C (Mines Jr. 2014). Solid tests are empirical in
character and very simple to perform due to the wide variety of inorganic and organic
materials encountered. Total suspended solids are the particles of more than 2.011m of
diameter that are retained by the filter. Total dissolved solids are found in potable
water in the form of inorganic salts, small amounts of organic matter, and dissolved
gases. The hardness of the water increases with total dissolved solids. Total suspended
solids (suspended colloidal and larger matter) increase with the degree of pollution.
Determination of total suspended and total dissolved solids is developed by the
measurement of filtered and unfiltered portions of samples (Sawyer, McCarty and
Parkin 2003).
Suspended solids cause aesthetic issues, a decline in aquatic species, and
ecological degradation of aquatic environment. Some of the effects of suspended
solids are the reduction of the amount of light penetrating into water which reduces
availability of energy for species survival (Bilotta and Brazier 2008). The typical
content of total solids in municipal wastewaters is approximately 720 mg L \ Total
suspended solids are between 100-350 mg L 1(vanLoon and Duffy 2011). The goal
of total suspended solids in wastewater treatments which is also defined by
regulations is below 30mg L 1(The Clean Water Act and NOM-003-
SEMARNAT-1997).
2.8.4 Total Nitrogen
Nitrogen components are contaminants of great concern because of the effects
that they have on the environment as well as on human health. Due to the extensive
use of fertilizers and pesticides, nitrogen components are found in wastewater in high
quantities. Due to the variety of oxidation states that nitrogen presents, there are many
nitrogen components. There are a total of seven oxidation states that result in the
21


formation of seven components, all of them are very important to the environment;
ammonia, nitrogen gas, dinitrogen monoxide, nitrogen monoxide, dinitrogen trioxide,
nitrogen dioxide, and dinitrogen pentoxide (Figure 2) (Doyle and Hoekstra 1981).
DINITROGEN PENTOXIDE
Figure 2 Seven Nitrogen Components
NH3, N23, and N25 mixed with water form inorganic ionized species;
ammonium, nitrite, and nitrate (Equations 5, 6 and 7), which are of environmental
concern in water and they are regulated (Bresler 2012).
nh3 + h2o 4 NH4+ + OH- (5)
N203 + H2C) 2H+ + 2NO
N205 + H2C) 2H+ + 2N3~
All nitrogen components can cause environmental problems except for N2
which is the main component of the atmosphere. The four forms of nitrogen that are
of interest in water resources are ammonia, nitrite, nitrate, and organic nitrogen.
22


Ammonia nitrogen represents all nitrogen that exists as an ammonium ion or
as ammonia. The nitrogen contained in organic compounds is the organic nitrogen.
Amino acids, nucleic acids, amines, amides, imides, and nitro derivatives are
examples of organic nitrogen components. Organic nitrogen components have little
significance in water analysis.
Nitrates and nitrites are nitrogen-oxygen chemical units mixed with organic
and inorganic compounds. Colorimetric procedures and ion chromatography are used
to determine nitrites due to the sensitivity required to measure them. Nitrate levels are
difficult to determine. Some of the methods used to determine nitrates are: ultraviolet
spectrometry, chromatographic and capillary ion electrophoresis, nitrate electrode,
and cadmium reduction (Senra-Ferreiro, et al.2010).
Total nitrogen (TN) is the sum of ammonia nitrogen, organic nitrogen,
nitrates, and nitrites. Gas nitrogen is not considered part of TN because of the
nonreactive effect that it has in water. TN gives a general idea or the nitrogen
component levels in wastewater.
Nitrogen components remaining in polluted waters are Indicators of Sanitary
Quality. When water contains mostly nitrate it means that water was polluted a long
time ago. This assumption is based on the oxidation sequence of the nitrogen
components. Typically, polluted waters contain organic nitrogen as the primary
pollutant. After some time, organic nitrogen is converted to ammonia nitrogen. If
aerobic conditions are present ammonia will be oxidized to nitrite and nitrate (Rossle
and Pretorius 2001). The typical content of TN in municipal wastewaters is
approximately 30-40 mg L (Taylor 2013). Nitrates are not usually present in
Municipal wastewater (Ahmed, et al.2012).
23


2.8.5 Fecal Coliform
Fecal coliform bacteria are microorganisms found in feces. They are
facultative anaerobes -they can survive in the absence of oxygen-, gram-negative,
non-spore forming, and they ferment lactose producing gas and acid when incubated
at 35C (Department of Environmental Sciences 2003). Although fecal coliforms are
usually not disease-causing organisms, they are tested to identify more harmful
present bacteria in the wastewater (Francy, et al.2004). Fecal coliform are indicators
of fecal pollution and of the presence of enteric pathogens of wastewater (Mack
1977). The test provides an easy and fast way to measure microorganism
concentration avoiding the use of additional tests to determine enteric viruses that are
difficult and time consuming. Fecal coliform is a good technique to determine fecal
contamination. It is also good as an indicator of pathogen regrowth because of the fact
that viruses and parasites cannot reproduce without a warm-blooded host (Efstratiu, et
al. 1988). However, the absence of fecal coliform does not mean that fecal
contamination is absent. Fecal coliform is also less reliable as an indicator of viruses
and parasites. Another disadvantage of fecal coliform is that during wastewater
treatment, some pathogens are more resistant than fecal coliform and they are not
readily removed. For example, viral pathogens have a greater survivability than fecal
coliform during anaerobic digestion. Thus, the measurement of fecal coliform after a
treatment may not provide accurate determination of all the pathogens (Department of
Environmental Sciences 2003). On the other hand, fecal coliform is a good indicator
of Salmonella sp. Fecal coliforms are the best predictors of the presence of
Salmonella sp (Efstratiu, et al.1988).
There are different methods to measure fecal coliform. One of the most
popular is the multiple-tube fermentation (MTF) which is performed using different
24


dilutions (Gronewold and Wolpert 2008). Fecal coliforms are identified by gas
production present in the Durham tubes. The average concentration of fecal coliform
in municipal wastewater is between 104 and 105 cells L'1. The FC concentration
should remain below 1.0E 03 100 mL'1 according to Garzon-Zuniga and Buelna
(2011).
2.9 Established standards and guidelines
Wastewater treatment and guidelines were not generally taken into
consideration for centuries. Wastewater was disposed of or discharged without any
treatment or regulation. Because of this, serious impacts on public health and the
environment were caused. The emergence of epidemics in Europe during the
nineteenth century was one of the critical effects of the incorrect disposal of the
wastes. In order to protect the health of the population and the environment, scientific
discoveries, debates on societal priorities, and government interest allowed the
establishment of standards and guidelines for the disposal and management of the
wastewater. The first Water pollution control regulation in the U.S. was put into
effect in the British colony of Massachusetts in 1647 (Lofrano and Brown 2010). The
major revolution in wastewater treatments and their regulation took place during the
20th century. The concepts of Biochemical Oxygen Demand (BOD5) and other
standards were presented in the Eighth Report (1912) of the Royal Commission on
Sewage Disposal. These standards were also applied in many other countries and they
began to mandate wastewater treatment. In 1950 after some debates of water quality
standards and stream use classification, the development of waste management
policies began.
In 1948 the US government, through Congress, enacted the first Federal Water
Pollution Control Act. The law did not provide enough regulations to control water
25


pollution and gave limited authority to the federal government. The law was then
continuously amended until 1972 when an appropriate legislation was obtained. The
Federal Water Pollution Control Act or Clean Water Act focuses on eliminating or
reducing the pollution of interstate waters and tributaries and improving the sanitary
condition of surface and underground waters.
Nowadays, the Environmental Protection Agency (EPA) is the primary
authority for the implementation of the Clean Water Act in the U.S. The EPA sets
effluent limits that apply to different water discharges to ensure the protection of the
receiving water bodies. The maximum national standards for secondary treatment in
the U.S. are 30 mg L 1 m BOD5, 30 mg L'1 in TSS and a range between 6 and 9 in pH
on an average 30-day concentration (Tchobanoglous, Burton and Stensel 2004). For
total nitrogen, the U.S. Environmental Protection Agency established a total nitrogen
range from 0.12 to 2.18 mg L'1 depending on the sensitivity of the region (Galil,
Malachi and Sheindorf 2009).
In Mexico, SEMARNAT (Secretary of Environment and Natural Resources) is
the principal authority determining wastewater parameters by the implementation of
the official regulation NOM-003-SEMARNAT-1997. There the maximum
permissible limits of pollutants in treated wastewater are 30 mg L 1 m BOD5, 30 mg
L_1 in TSS,1.E + 03.100 ml in FC and a range between 6 and 9 in pH
(SEMARNAT 1997). For total nitrogen the NOM-001-SEMARNAT-1996
established 15 mg L'1 as the maximum permissible limit for the aquatic wildlife
protection.
26


2.10 Previous studies
2.10.1 Organic biofilters
Wastewater treatments using bio-filters packed with organic materials have
shown efficiency in pollutant removal. Some of the biological residuals used for
packing material, also called filter media, are agave, compost, peat, soil, wood shell,
wood chips, heather, as well as coconut, date-palm, and bamboo fibers (Vigueras-
Cortes, et al.2013; Lens, et al.1994; Buelna and Belanger 1990; Garzon-Zuniga and
Buelna 2011; Gao, et al.2011; Nicolai and Janni 2001; Riahi, Mammou and Thayer
2009). The biofilter should have a porous solid media that allows the support of
microorganisms and the access of pollutants in the airflow (Nicolai and Janni 2001).
Thus, pollutants are removed through degradation with microorganisms. Biological
residuals such as date-palm fibers are natural, inexpensive, and environmental
friendly materials that can be used toward biofiltration (Riahi, Mammou and Thayer
2009).
There have been many studies that used different types of packing materials.
Riahi, Mammou and Thayer performed an experiment using columns packed with
Phoenix dactylifera one of the most cultivated palms around the world. Date-palm
fibers were chosen because of their wide waste availability as well as large amounts
of waste after the trimming operations. Garzon-Zuniga and Buelna (2011) developed
a trickling biofilter (TBF) that was packed with a blend of priming waste from two
tree species; Dwarf Poinciana (Caesalpina pulcherrima) and Jacaranda (Jacaranda
mimosifolia) which are ornamental trees obtaining removal efficiencies of 97% in
BOD5, 71% in COD, 95% in TSS and 4 log units in fecal coliforms.
Organic biofilters are an efficient and economical method to decrease
turbidity, phosphorous, chemical oxygen demand (COD), and helmith eggs of
27


secondary domestic water. Organic media provides adequate pollutant removal
because it allows the microorganisms growth. Thus, biomass accumulation is
possible inside of the reactors and the biodegradation of the pollutants is improved.
There are also some experiments that did not use organic material at all. Rebah et al.
(2010) developed two different biofiltration systems using clay granular media in one
of the systems and plastic media in the other. Rebah developed aerobic and anaerobic
scenarios in each system in order to provide the needed conditions to the nitrification
and denitrification processes. Recently, Vigueras-Cortes et al.(2013) developed two
series of bio-filters using agave fiber in the municipal wastewater treatment process.
Agave fiber waste is generated during mescal production. Agave fiber is a
solid waste inexpensive and environmentally friendly. It also has a wide contact area
which facilitates biofilm formation that degrades wastewater pollutants. They
evaluated laboratory scale biofiltration reactors in two trials, installing aerobic and
anaerobic bioreactors. They used five different hydraulic loading rates (HLRs) of
0.27, 0.54, 0.80, 1.07, 1.34 m3 m~2 d_1 obtaining removal efficiencies of 92% in
BOD5, 79% in COD, 99.9% in fecal coliforms and 91% in TSS.
BFs have been developed using different column materials and different
dimensions. For example, Rebah et al.(2010) used Plexiglass columns of 2-1.4 m by
0.2 m. Riahi et al. (2009) set up three pilot scale reactors with a 0.55 m long glass
column and 0.06 m of internal diameter. Vigueras-Cortes et al.(2013) made each BF
from PVC (Polyvinyl Chloride) pipe of 2 m tall and 0.185 m of internal diameter. The
bio-filtration column packed with agave fiber was 1.80 m tall.
The characteristics of the influent and its flow rate play an important role in
the performance of the filter. The inffluent of Riahi et al. (2009) was introduced into
the reactors in a constant flow rate ranging from 11.8 to 72 mL min"1 using domestic
28


wastewater from the secondary effluent of an activated sludge treatment plant.
Vigueras-Cortes et al.(2013) used wastewater flow rates ranging from 5 to 25 mL
min'1 with wastewater collected after primary treatment of a treatment plant.
2.10.2 The start-up period
The start-up period is a fact that determines the performance of the biofilters.
It is the period of time to grow the biofilm, provided to wastewater treatment in order
to stabilize and routinely control the process (EPA 1973). Rebah, et al.2010, with the
clay and plastic media systems, provided a 2 week start-up period to the systems. But
some other studies provided even one or two months. Vigueras-Cortes, et al.2013
provided 3 months at a flow rate of 3 mL min'1 for the agave BFs to be conditioned.
During the phase right after the start up, sometimes the COD and other pollutant
removal were not very efficient. This could be explained due the lack of
acclimatization of the biomass in the reactor. When this happened, the initial phase
forms part of the start-up (Rebah, et al.2010).
2.10.3 Studies that remove nitrogen effectively
There are some packing materials that have been shown efficient in the
removal of nitrogen. Providing aerobic-anaerobic scenarios to obtain nitrification-
denitrification processes have recently been applied to the nitrogen removal. During
the nitrogen cycle, three stages are developed; aerobic nitrification, anaerobic
denitrification, and nitrogen fixation (Bemat, et al.2011). Thus, systems with aerobic
and anaerobic stages can achieve nitrification-denitrification processes. Removal of
nitrogen through nitrification-denitrification has been proven in previous research.
Many studies used different methods and/or media to develop nitrification-
denitrification processes by the establishment of aerobic-anaerobic stages.
29


Bemat et al.(2011) developed a sequencing batch reactor (SBR) including
aerobic and anaerobic phases in the modified cycle. Bemat objective was to evaluate
nitrification and denitrification processes at low DO concentration. Papers such as
Nozhevnikova, et al.2012 relates the efficiency of nitrogen removal to the conjugated
nitrification, denitrification, and anammox processes. The functioning of nitrification,
denitrification, and anammox processes was demonstrated during laboratory
cultivation methods and during previous studies of the processes in batch and
continuous reactors. Yuan, et al. 2012 developed a step-feed anaerobic-(oxic/anoxic)
Membrane Bioreactor. The complete system consisted of an anaerobic reactor and
then multiple phases of aerobic and anoxic zones in sequence, concluding with a
continuous aerated membrane bioreactor. However, only few studies of nitrogen
removal using aerobic reactors in series have been carried out.
2.10.4 Anaerobic fitters
Anaerobic pretreatments are used to break down biodegradable substances and
to decrease organic material. Besides pollution reduction, anaerobic treatments
produce energy through methanogenesis. Low sludge production has been achieved in
established anaerobic wastewater treatments. In addition, anaerobic treatments do not
require a complicated operation nor high operation costs (Rebah, et al. 2010) because
no oxygen is needed. The combination of filter media with anaerobic conditions
results in total biomass retention, excellent effluent quality, low sludge production,
small foot print, and energy production (Lin, et al.2013). COD depletion has also
been observed in anaerobic conditions (Zeng, et al.2010).
2.10.5 Aerobic filters
In order to provide aerobic conditions, air is injected to the bio-filters. The air
is usually injected at the bottom of the BF. The aeration rate may be different for each
30


experiment. There are some studies that just maintain the air concentration at some
level introducing the sufficient amounts of air. Vigueras-Cortes et al.(2013) used
mechanical aeration by injecting air at the bottom of three biofilters of six. The
aeration rate was 0.62 m3 m'2 h'1.
Nitrification-denitrification processes as well as phosphorous removal in high
levels have been achieved using only aerobic bio-filtration (Rebah, et al.2010).
Bemat et al. (2011) achieved ammonia removal using volumetric exchange rate of 0.1
d-1,0.3 d-1, and 0.5 d_1 during the aeration phases observing ammonia oxidation
through zero order kinetics. The ammonia nitrogen oxidation achieved at the end of
the phases was from 96 to 98.4%. DO level is one of the key factors when developing
nitrification-denitrification (Zeng, et al.2010).
2AO. 6 Other factors influencing aerobic and anaerobic biofilters
Temperature: The temperature of the biofilters can be used as a co-variable of
their operation. Rebah et al. (2010) operated their aerobic BFs under room
temperature while in the anaerobic BFs the temperature was fixed to 35C. Energy
must be provided in order to maintain temperatures above room temperature. Thus,
operation costs increase. Vigueras-Cortes et al.(2013) operated the BFs under their
natural temperature without providing any energy that would increase operation costs.
pH: A neutral pH seems to be suitable for nitrification and denitrification
processes in BFs. Zeng, et al.(2010) reported a pH range between 7.12 and 7.46 in
the nitritation and denitritation of domestic wastewater. During their study, Vigueras-
Cortes et al.(2013) observed a neutral pH between 6.67 and 7.28 in every step of the
process.
31


2.10.7 Nitrification and denitrification processes using anaerobic and aerobic
systems
Many wastewater treatments have applied anaerobic-aerobic process to
eliminate nitrogen and other pollutants. Some examples are anaerobic filter-activated
sludge system, up-flow anaerobic sludge blanked (UASB)-attached aerobic filter,
anaerobic baffled reactor-activated sludge system, and aerobic-anaerobic filters
(Rebah, et al.2010). Nitrogen removal has been attributed to the complete
nitrification process and denitrification phenomenon that occurred (Garzon-Zuniga
and Buelna 2011). During nitrification, ammonia is oxidized forming nitrates by
Nitrobacteria action and nitrites by Nitrosomas action. Thus, existing predominant
bacteria determine the majority of the nitrogen form (nitrates or nitrites). When
concentration of nitrites is higher than concentration of nitrates, an incomplete
nitrification process was performed (Rebah, et al. 2010). To achieve nitrogen
removal of wastewater, a partial nitrification to nitrite and then to nitrate must be
developed, followed b denitrification (Bemat, et al. 2011). Denitrification is enhanced
because of the presence of anoxic zones that allowed the growth of heterotrophic
bacteria that reduce nitrates to nitrogen gas (Galil, Malachi and Sheindorf 2009).
Highly concentrated wastewater can effectively be treated using combined anaerobic-
aerobic processes (Lin, et al.2014). A start-up period is necessary to obtain excellent
organic removal.
Rebah et al. (2010) developed two anaerobic-aerobic biofiltration systems
using clay (system 1)and plastic media (system 2) as packing material. Each system
had two reactors connected in series. Wastewater was first pumped to the anaerobic
reactor. The effluent of the anaerobic reactor was then introduced to the aerobic one
as the influent in order to remove remaining contaminants; they were operated as plug
32


flow reactors. They obtained removal efficiencies of 91-98% in BOD, 90% in COD,
and 60-70% in nitrogen removal. Therefore, the performance of the aerobic reactor
was determined based on the performance of the anaerobic reactor which constitutes
the first part of the treatment (Lin, et al.2014).
33


CHAPTER III
METHODS
This section reviews in detail the methods used for this study. Specifically, I
outline how the anaerobic-aerobic system functions, as well as the packing material
used. More precise discussions follow, surrounding the topics of wastewater influent,
the start-up period, and experimental procedure before briefly summarizing the
analytical methods and experimental design used.
3.1 Hybrid system
The system included two reactors connected in series, an anaerobic reactor
followed by an aerobic performed in duplicate. Each system was operated as plug
flow reactors (Vigueras-Cortes, et al.2013). Figure 3 shows how the raw wastewater
was first pumped to the top of the anaerobic reactors (BF1 and BF2) with peristaltic
pumps MasterFlex Model 751800. After the treatment in the anaerobic reactor, the
effluent was fed into the aerobic reactors (BF3 and BF4) to finish with the treatment.
ANAEROBIC AEROBIC
FILTERS FILTERS
Influentl Influent 2
Figure 3 Schematic diagrams of the two combined anaerobic-aerobic BF systems
34


Figure 4 Anaerobic-aerobic BF system
The reactors consisted of polyvinyl chloride (PVC) pipe, 2.0 m high with an
internal diameter of 0.185 m. The four biofilters (BFs) were packed with agave fiber
that was previously washed and dried according to Garzon-Zuniga et al.(2003). The
biofilters had a 1.80m biofiltration column packed with agave fiber. Additionally, four
plates were located along each column as internal divisions in order to separate the
packing material, to prevent compaction, and to avoid plugging or clogging of the
fiber. The acrylic plates were perforated to allow wastewater flow and the system was
run at room temperature.
The aerobic BFs were supplied with an air loading of 0.62 m3 m'2 h'1 counter
current flow of wastewater. The air supply was monitored daily using a Dwyer RMA-
2 flow meter. Pressure drop was also measured using a pipe connected to a hydraulic
gauge. The BFs were installed outdoors in order to mimic a natural environment of a
possible future installation in the rural municipalities of Durango. The temperature of
_SR
the BFs was used as a co-variable.
35


3.2 Filter material
Agave plant has an extensive use in Mexico. It is used in the production of
medicine and fibers as well as in fermented and distilled drinks. There are around 48
species of agave used in spirits production (Ramirez-Tobias, et al.2012). Agave is a
species that can be located in arid and semi-arid regions in the north of Mexico. The
species Agave durangensis is cultivated in Durango (Figure 5). This plant is used on
Mescal production, the typical distilled spirit of the region that is an alcoholic
beverage similar to tequila. In Durango, 3.8 million agave plants were cultivated in
27 according to the statistics of SEDECO (2008). After mescal production by
fermentation and distillation of the plant, the agave fiber is considered useless
material and is disposed.
Figure 5 Agave Duranguensis
Plants from arid and semi-arid regions such as agave can provide a wide
variety of organic materials to the treatment of wastewater. This represents an
advantage to these regions due to the scarcity of water and the necessity of water
36


reuse. Agave plants have many advantages; they are able to adapt to dry climates by
storing water and growing slow. Agave fiber is an organic material readily available,
inexpensive, and is also considered a waste of the alcoholic beverage processes
(Vigueras-Cortes, et al.2013).
Agave fiber wastes can be used to pack columns in the biofiltration of
wastewater. In addition to filtration, this material favors the retention of organic
matter and pollutants by adsorption, absorption and ion exchange mechanisms
(Garzon-Zuniga, Tomasini-Ortiz, et al.2008). Thus, agave fiber represents a potential
organic material in the development of BFs to the treatment of municipal wastewater.
This experiment employed agave fiber from the mescal industry in Nombre de
Dios, Durango. The fiber was transported to the research center (Figure 6), washed
and dried outside (Figure 8). It was also passed through a 100-mesh sieve removing
small particles.
Figure 6 Agave fiber reception
37


Figure 7 Agave fiber before washing
Figure 8 Preparation of the agave fiber
The fiber was analyzed by measuring cellulose, lignin and acid detergent fiber
concentrations. This characterization was made using the methods described by
Tejada (1985). The aparent density and porosity were also measured from a small
volume of fiber. Four columns were packed in the compartment between the four
perforated acrylic plates contained. The packing was based on the moisture content of
65% that was previously wetted with tap water. These measurements and procedures
38


of the packing material were based on the ones presented by Vigueras-Cortes, et al.
(2013) and the references cited therein.
Influent
Figure 9 Biofilter single column
3.3 Wastewater influent
The raw wastewater was collected once every week from the Municipal
wastewater treatment plant of Durango City. The wastewater was sampled from the
plant after the primary treatment, which provides screening, sedimentation, and
homogenization. The sample of wastewater was taken during the first peak mass-
loading (Figure 10). The peak mass-loading refers to the period of time when
wastewater concentration is the highest. For Durango City, the first peak mass-
loading is between the 12th and 15th hour each day. At this time, the plant receives all
the wastes generated during the morning activities. The second peak mass-loading is
at the 18th to 20th hour. However, the collection of the wastewater was only
developed during the first peak.
39


Figure 10 Wastewater collection
The wastewater collected was then transported to the laboratory and properly
disposed in a 400 L polyethylene tank. The wastewater was fed in the BFs with
peristaltic pumps (MasterFlex Model 751800).
3.4 Start-up period
The start-up period was provided to the BFs after leak and nole tests on the
filter material. During this time, the BFs were evaluated for three months using a flow
rate of 5 mL min'1. The first step ended when the parameters of BOD reach the
allowable values for the standard regulations (30 mg L'1) and remain stable. No other
seed was used for the start-up phase of the BFs.
3.5 Experimental procedure
After the start-up period, the flow rate of the effluent was increased. Four
different flow rates were tested;10,15, 20, and 25 mL min'1 that correspond to four
different HRL (Hydraulic loading rates); 0.54, 0.80, 1.07, and 1.34 m3 m'2 d_1,
respectively. The HRL indicated the four phases of the experiment. As it was
mentioned, the aeration rate in the aerobic filters was 0.62 m3 m 2 h \ The aerobic
40


filters were labeled BF1 and BF2. The non-aerated BFs were labeled BF3 and BF4
(Figure 11).This experiment was conducted within 360 days continuous.
3.6 Analytical methods
The following parameters were analyzed according to the Standard Methods
(APHA 1998). Biological oxygen demand according to the 5-days BOD5 test method
5210; Chemical Oxygen Demand according to the Closed Reflux, Colorimetric
method 5220; Total Solids according to Total Solids Dried at 103-105C method
2540; Fecal Coliform according to Fecal Coliform Procedure method 9221; Nitrates
41


by capillary electrophoresis with ultraviolet detector to 211 nm using a buffer 20
mmol Tris and 20 mmol NaCl.
Samples from the influent and effluent of each system and each BF were
analyzed every week (Figures 13). Electrical conductivity (EC) and pH were also
measured once a week. Electrical conductivity was measured according to
Conductivity, Laboratory method 2510 also from APHA, 1998. Air flow rate internal
and room temperature were monitored from Monday to Friday.
Figure 12 Effluents of the start-up period
Figure 13 Effluents and influent of the last phase
42


3.7 Experimental design
Two variables were considered in the experimental design based on the HLRs
(0.54, 0.80, 1.07, and 1.34 m3 m~2 d-1) and the aeration (with air and without air). The
variables were tested using a factorial experiment. Internal temperatures of the BFs
were co-variables in the experiment. The statistical method and its sources of
variation were evaluated with the Statistica software (StatSoft, 2004).
43


CHAPTER IV
RESULTS AND ANALYSIS
This section outlines the results obtained in the different parameters of the
study. First, I include a table with the wastewater characterization with the initial
concentrations in the influents. Then, final concentrations and removal efficiencies of
organic matter, fecal coliforms, total suspended solids, and nitrates are concisely
presented and illustrated. Additional graphs are also presented to illustrate some of the
statistical analyses.
4.1 Wastewater characterization
The composition of the raw wastewater that was used in the study is shown in
table 4.1.
Table 4.1 Average composition of the raw wastewater
Parameters Average concentration S.D. Units Sample number
Biochemical Oxygen Demand 252 37 mgL'1 27
Chemical Oxygen Demand 706 165 mgL'1 29
Total Suspended Solids 62 36 mgL'1 19
Fecal Coliform 1.26x1072.15x107 MPN 100 mL_1 35
Nitrate 0.0 mgL'1 38
Electric Conductivity <850 |xS cm-1 31
pH 7.07 0.69 30
S.D. = standard deviation, MPN= most probable number
4.2 Organic matter (BOD5 and COD) removal effect
During the start-up period (HRL=0.27 m3 m~2 d-1), removal of BOD5 occurred
in the aerobic reactors achieving concentrations below the allowable 30 mg L'1
44


(Figure 15). The stat-up period was eighty five days and ended once BOD5
concentration in the effluents achieved levels in compliance with the regulations and
was constant. However, the anaerobic reactors did not achieve the permissible levels
and they have an increase in BOD5 concentration up to 150 mg L'1 during the start-up
period (Figure 14). These results match with the statistical analysis which indicates
that aerobic BFs provide the highest BOD5 removal.
During the first stage (HLR=0.54 m3m~2d~1), effluents from anaerobic reactors
were slightly above the regulation but once the aerobic treatment was provided,
concentrations below 30 mg L'1 were always achieved. The following two stages of
the BFs (HLR=0.80 and 1.07 irfm-M"1) were also able to provide permissible
concentrations after the aerobic treatment. The maximum removal efficiency of BOD5
in the system was 96% with an effluent of only 17 mg L'1.
Figure 14 Effluent BOD5 concentrations in the anaerobic BFs operated at four
different hydraulic loading rates
45


400 i
250 -
200 -
150 -
100 -

50
1.34 m3/m2 d
50
100
150
200
250
300
350
-A-BF air x Influent Time (d)
Figure 15 Effluent BOD5 concentrations in the aerobic BFs operated at four different
hydraulic loading rates.
The COD had an increase during the start-up period in the anaerobic BFs,
reaching a COD concentration of almost 2000 mg L'1(Figure 17). This might be
caused by the dissolution or washing of the caramelized carbohydrates of the fiber
that were attached during the cooking process of the mescal production. On the other
hand, the aerobic BFs started removing COD from the start-up period (Figure 18).
The COD concentrations in the effluents decrease from 750 to 400 mg L'1 during the
start-up period of the aerobic BFs which indicates that aeration promotes fast removal
of components. In the first stage (HLR=0.54 irfm-M"1)the COD decreased
significantly maintaining COD concentrations below the influent concentrations.
During the first stage COD concentrations decreased from 1080 to 250 mg L'1 and
from 380 to 130 mg L'1 in the anaerobic and aerobic BFs, respectively.
The maximum removal efficiency of COD was 87% at HLR of 1.34 m3m-2d-1
in the aerobic BF. Thus, in the aerated BFs, the COD had significant reductions. The

0 0
5 0
3 3
h-E^aoa
46


lowest concentration of COD was 50 mg L'1 and was observed at the end of the
2500
0.80 m3/m2d
1.07 m3/m2d
1.34 m3/m2d
1500
1000 -
1000
500
0 50 100 150 200 250 300 350
-e-BF without air x Influent Time (d)
Figure 16 COD average concentrations in the anaerobic biofilters
experiment (HLR=1.34 m3 m~2d-1).
2500
0.27 m3/m2d
0.54 m3/m2 d
0.27 m3/m2 d 0.54 m3/m2 d 0.80 m3/m2 d 1.07 m3/m2 d
n >
( V X

> x / >* X

0 50 100 150 200 250 300 350
-A-BF air x Influent Time (d)
Figure 17 COD average concentrations in the aerobic biofilters
o o
o o
o 5
2 1
(1/6Ea0
o
o
(1/6E) a0
47


4.2.1 Statistical analysis of the organic matter removal efficiency
4.2.1.1 Biochemical Oxygen Demand
According to the covariance test the temperature, air and air^HLR interaction
were statistically significant (p=0.001) for BOD5 removal. Mean analysis based on the
Fishers method showed that the hybrid system had more BOD5 removal efficiency at
HLR of 0.54 m3 m-2d-1 and the BFs with air effect had more efficiency (Figure 16).
HLS (m3m-2d-1) '
Figure 18 Air*HLR interaction on biochemical oxygen demand removal in the
system at four different hydraulic loading rates.
Table 4.2 BOD average concentrations in the effluents
HSL (mW1) BF w/out air (mg L1) S.D. BF air (mg L1) S.D.
0.54 44 27 17 5
0.8 47 12 32 18
1.07 21 16 39 18
1.34 57 17 57 18
S.D. = standard deviation

o o o o o o
2 0 8 6 4 2
(_l/o)E)lnclg
48


4.2.1.2 Chemical Oxygen Demand
According to the covariance test the intercept, temperature and air were
statistically significant (p=0.001) for COD removal. Mean analysis based on the
Fishers method showed that the hybrid system had more COD removal efficiency at
HLR of 1.34 m3 m~2 d-1(Figure 20) and the BFs with air effect had the highest
removal efficiency (Figure 19).
Air Without air
Figure 19 Air effect in the Statistical analyses of COD removal in the hybrid system
Table 4.3 COD average concentrations in the effluents
HSL (mW1) BF w/out air (mg L1) S.D. BF air (mg L1) S.D.
0.54 530 262 299 124
0.80 337 92 129 43
1.07 349 70 130 40
1.34 286 88 128 86
S.D. = standard deviation
o o
o 5
3 2
(LraE) ao
49


450
Figure 20 HSL effect in COD removal in the hybrid system
4.3 Fecal coliforms removal effect
The hybrid system showed removal of FC even during the start-up period.
During the first and last stages (HLR=0.54 and 1.34 m3 m"2d-1)FC removal
efficiencies reached 98 (1log units) and 99.98% (3 log units) in the unaerated and
aerated BFs, respectively (Table 4.4). The best stage for FC removal was in the
aerobic BFs at HLR of 0.80 m3 m-2 d-1(Figure 21), the maxima average FC removal
efficiency from the effluent reached 99.8 (2 log units) and 99.99% (4 log units) in the
unaerated and aerated BFs (Table 4.4), respectively. When the HLR was 1.07 m3
m"2d-1 the FC removal efficiency was 99.98% (3 log units) in both aerobic and
anaerobic BFs. FC removal in the start-up period was lower than in the first stages.
50


1.00E+08
1.00E+07
E
o

^ 1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
Figure 21 Fecal Coliform concentrations in anaerobic biofilters
Figure 22 Fecal Coliform concentrations in aerobic biofilters
IE 00'NdESEJOioo Isad
51


Table 4.4 Fecal coliform removal efficiencies in logarithmic units at four different
hydraulic loading rates
HLR (m3m 2d BF w/out air (log units) BF air (log units) BF w/out air Removal % BF air Removal %
0.54 1 3 98.83 99.98
0.8 2 4 99.83 99.99
1.07 3 3 99.98 99.98
1.34 1 3 98.83 99.98
43A Statistical analyses of fecal coliform removal
According to the covariance test, the intercept, air, hydraulic loading rate, and
air*HLR interaction were statistically significant (p=0.001)for fecal coliform
removal. Mean analysis based on the Fishers method showed that the hybrid system
had more Fecal Coliform removal efficiency at HLR of 1.34 m3 m-2d-1 (Figure 24)
and that the air effect provided the highest removal efficiencies (Figure 23).
Figure 23 Air effect in the Statistical analyses of FC removal in the hybrid system
52


4E5
3.5E5
O'
E 3E5
0
| 2.5E5
CL
1 2E5
£
^ 1.5E5
O
| 1E5
50000
0
0.54
0.80 1.07
HSL (m3m-2d-1)
.34
Figure 24 HLR effect ot lecal coliform removal in the hybrid system
4.4 Total suspended solids removal effect
Total suspended solids concentrations of the effluents are shown in figure 23.
The highest TSS removal efficiencies were observed when HLR was 1.07
but the lowest TSS average concentration was observed at HLR of 0.80 m3 m~2 d-1
(Table 4.5). They were 80 and 77% in the unaerated and aerated BFs, respectively.
However, in the aerobic BFs the TSS removal efficiency was 78% when HRL was
0.54 m3 m_2d-1 (Table 4.5).
Table 4.5 Total suspended solid effluent concentrations and removal efriciencies
HLR (mW1) BF w/out air Effluent concentration (mg L1) S.D. BF w/out air Removal (%) BF air Effluent concentration (mg L1) S.D. BF air Removal (%)
0.54 35 28 47 14 8 78
0.8 22 16 39 13 9 63
1.07 15 8 80 17 7 77
1.34 17 4 75 18 7 73
S.D. = standard deviation
53


NOM-003-SEMARNAT-
20 -
0 50 100 150 200 256 300 350
BF air aBF without air x Influent ' ^ >
Figure 25 Total suspended solids concentrations in the two biofilter series
4.5 Nitrates removal effect
Two hybrid systems were tested for N3_ removal. System 1(wastewater flow
from BF1 to BF3) had a- more unstable behavior concerned to nitrates removal.
System 2 (wastewater flow from BF2 to BF4) provide the best performance in nitrates
removal at HLR of 0.54 and 0.8 m3 m-M"1. Results for nitrates removal (N3_) in
system 2 are presented in figure 26.
The lowest level of NO3" concentration observed in the systems was an
average of 7 mg L_1 at HLR of 0.54 m3m-2d-1 (Table 4.6). The anaerobic BF of
system 2 had NO3" effluent concentrations below 10 mg L'1 at HRLs of 0.54 and 0.80
m3 m-2 d-1(Figure 26).
00000000
86420864
(1/600 sfD-os,1,fDuedsns lelol
54


350 n
Mean analysis based on the Fishery method showed that anaerobic BFs had
less N03- production at HLR of 0.54 irfm-M.1 (Table 4.7). The intercept, air,
temperature and HLR were statistically significant for N03- removal.
Table 4.6 N03- average concentrations in the effluents
HSL (m3 m^d-1) BF w/out air (mg L-1) S.D. BF air (mg L1) S.D.
0.54 7 3 135 38
0.80 10 5 211 76
1.07 11 2 179 46
1.34 17 8 222 9
S.D. = standard deviation
4.5.1 Statistical analysis of the NOs~ removal
According to the covariance test the intercept, air, hydraulic loading rate and
HLR interaction were statistically significant (p=0.001)for N03_ removal. Mean
55


analysis based on the Fishers method showed that anaerobic BFs (Figure 27) had
Figure 27 Air effect in the statistical analysis of N03- removal in the hybrid system
180
160
40
20
0.54
0.80 1.07
-21
1.34
Figure 28 HLR effect of N03- removal in the hybrid system
more N03" removal efficiency at HLR of 0.54 (Figure 28).
(1/6E) VON
SEroN
56


CHAPTER V
DISCUSSION
This section presents a detailed discussion of the results obtained for this
study. Specifically, I review the results of each parameter and compare them with
other studies. Additionally, possible explanations of some behaviors are explained
based on literature.
5.1 Organic matter (BOD5 and COD) removal effect
In the BFs without air, the majority of the BOD concentrations were not
allowable by the Mexican regulations. However, the aerobic treatment in the aerated
BFs was highly successful in the removal of BOD. The HLRs from the start-up period
to the second stage (0.80 m3 m~2 d-1) provided results below 30 mg/L of BOD.
Low BOD removal in the anaerobic BFs at the beginning of the experiment
can be explained because of the low development of anaerobic biofilm present at the
first stage as well as the effect caused by the washing deficient of the packing
material. However, the allowable concentration of BOD was not achieved during the
anaerobic stage.
The agave fiber BFs obtained lower concentrations of BOD than Rebah, et al.
(2010) that obtained concentrations between 42 and 74 mg L-1 treating synthetic
wastewater within two hybrid systems using a secondary sludge from a municipal
wastewater treatment plant and temperature of 35 C.
The agave fiber BFs achieved similar removal efficiencies to Garzon-Zuniga
and Buelna (2011) that obtained a removal efficency of 97% of BOD using priming
waste from two tree species, mechanical aeration, and an average influent of 264 mg
L_1. But, the HLR of the experiment was almost 10 times smaller (0.078 m3 m-2d-1)
than the HLRs used in this study (0.54 to 1.34 m3 m"2d-1).
57


Comparing BFs with the same structure and organic media, Vigueras-Cortes,
et al.(2013) obtained a BOD of <30 mg L-1 in the columns without air when using
HLRs of 0.54 and 0.80 m3 m-2 d-1 and in the columns with air when using HLRs from
0.27 to 0.80 m3 m~2 d-1. The variation in the results can be awarded to the prewashing
and predrying that was provided to the fiber in this study.
One of the studies that obtained higher removal efficiencies than agave fiber BFs
was Gilbert, et al.(28) that obtained BOD removal 95-99% using a three layer
biofiltration of 1)pozzolana, 2) mix of wood chips and peat, and 3) mix of peat, wood
chips, and cal cite. However, the HLR used was very small, 0.017 m3 m"2d-1.
Garzon-Zuniga (2011) obtained 99% removal of BOD treating wastewater from
pig farm using chip wood and peat as the filter media and HLRs of 0.07, 0.045, and
0.035 m3 m~2 d-1. Buelna, Garzon-Zuniga and Moeller-Chavez (2011) obtained 97%
of BOD removal using an HLR of 0.78 m3 m-2 d-1. This BF showed obstruction
problems when working with high HRL. The lack of obstruction problems with high
HRL is an advantage for the agave fiber BFs.
5.2 Fecal coliform removal effect
The FC removal efnciencies of the aerated BFs met the Mexican and US
standards (1,000 MPN/100 mL-1) in few cases at HLRs of 0.54-1.07 m3 m-2d-1 in the
aerobic BFs. The aerated BFs at HLRs of 0.54 and 1.07 m3 m"2d-1 achieved the
maxima average FC removal efficiencies of 99.95% (3 log units). The unaerated BFs
did meet neither the Mexican nor US regulations in FC. However, the effluents of the
anaerobic BFs were always introduced to the aerobic ones, so few of the final
effluents were able to meet the regulations. FC removal in the start-up period was
lower than in the first stages. According to Garzon-Zuniga and Buelna 2011 the FC
58


removal efficiency starts occurring increasingly once the filtering medium is
saturated. Other mechanisms might affect such as predation by testates amoebae.
Although good FC removal efficiencies were achieved, the effluents must be
disinfected before being used on irrigation or other water reuse activity. However, to
avoid this process is recommended to provide a tertiary treatment such as polish
lagoon, uv radiations or ozone. Nevertheless, these FC removal efficiencies are
greater than the efficiencies reported by Tremblay, Lessard and Lavoie (1996) that
used a trickling BF and obtain only 56% reduction of FC. They are also greater than
the efficiencies reported by Han and Dague (1997) that obtained 66% of coliform
reduction in a mesophilic reactor. Additionally, there are some authors that did not
achieve FC removal such as Shah, et al.(2002) that worked with a wheat straw BF
and treated dairy wastewater. Shah, et al.experimen resulted in an increase of FC
from 504xlO6 cfu/lOOmL in the influent to 665xl06 cfu/lOOmL in the effluent.
The removal efficiencies of FC in the agave fiber BFs were similar to Garzon-
Zuniga and Buelna 2011(4 log units) with an HLR of 0.078 m3 m"2d-1, approximately
a tenth of the HLR of the present study. Hill, et al.(2002) also obtained FC removal
efficiencies from 97 to 99% with water temperatures between 23 and 32 C and
providing UV radiation. The agave BF system did not require other resource more
than air injection. This represent and advantage compared with other systems.
5.3 Total suspended solids (TSS) removal effect
The TSS removal efficiency was better in anaerobic BFs (80%) than in aerobic
ones (78%). The high percentage of voids in the filter material is related with the TSS
removal efficiency of the BFs and allows for efficient retention of TSS (Vigueras-
Cortes, et al.2013). The TSS removal efficiencies (77-80%) are low compared with
the efficiencies of other parameters, however, the concentration of TSS in the influent
59


was actually low because of the efficient primary treatment that is provided to the
water in the wastewater treatment plant (WWTP). Despite the low efficiencies
removal,TSS in the effluent of the aerobic BFs meet the allowable levels in the
NOM-003-SEMARNAT-1997 and the EPA regulations (30 mg L_1 of TSS). The TSS
removal efficiency was greater than the one obtained by Lens, et al.(1994) that
obtained only 72% using peat, bark and wood chips as a filter media.
Garzon-Zuniga and Buelna (2011) obtained a TSS removal efficiency of 95%
with no mechanical aeration and with a HLR 10 times smaller than the hybrid system.
Vigueras-Cortes, et al.(2013) obtained 93.4 and 91.9% of TSS removal efficiency in
the anaerobic and aerobic BFs, respectively using almost the same conditions that in
this study but no hybrid connection between the BFs. However, the TSS concentration
in the raw wastewater of Vigueras-Cortes (201 67 mg L_1) was higher than the
concentration of the wastewater used in this study (62 36 mg L_1). Thus, the final
concentrations of TSS were low and in compliance with the regulations.
5.4 Nitrates removal effect
The highest removal efficiency of N03- was observed m system 2 (from BF2
to BF4) in the majority of the stages. The concentrations of N03- in the effluents of
the BF1 and BF2 decreased because of the denitrification that occurred in the reactors.
Consequently, the concentrations of N03- increased in the effluents of BF3 and BF4
due to the nitrification of ammonia-nitrogen. Denitrification occurred when anaerobic
conditions were provided to the microorganisms, as pointed out by Busigny, et al.
(2013), and not in aerobic conditions by the heterotrophic bacteria as pointed out by
Wen and Wei (2011). Thus, low concentrations of NO-3 were obtained because in the
influent the majority of nitrogen compounds were NH4 and nitrification was slightly
performed. However, the anaerobic stage helped the system to degrade organic
60


material, and the lowest concentrations of nitrates that were obtained in the BFs were
7 mg L'1. These N03- concentrations are lower than the 110,130, and 85 mg L'1
measurements obtained by Bemat, et al.(2011) and were obtained by treating a
mixture of wastewater in an SBR (Sequencing Batch Reactor) at limited oxygen
concentration and with anaerobic sludge digester supernatant. The N03-
concentrations are higher than the ones obtained by Galil, Malachi and Sheindorf
(2009) in their anaerobic reactor (between 4.8 and 6.2 mg L'1) but in their case, they
recycled sludge from an anoxic reactor that they implemented. The obtention of low
nitrate concentrations also reflects that in the anaerobic reactors there may be some
aerobic spots where nitrification has taken due to the lack of complete hermeticity of
the BFs.
Nitrification of ammonia-nitrogen was performed in the aerobic BFs. This
assumption is evident because of the high production of nitrates in the aerobic BFs.
The concentrations of nitrates in the aerobic BFs were between 135 and 222 mg L 1 m
average. Thus, the ammonia remaining from the anaerobic step was transformed in
the aerobic step. However, final concentrations of NH4 are needed in order to prove
the removal of this component.
Li, et al. (2013) also had an increment of N03- concentrations at the end of the
treatment where wastewater was treated with an SBR that included with 4 tanks of
different anaerobic/anoxic/aerobic function. This increased N03- concentrations from
2% in the influent to 47% in the effluent of the total nitrogen, most likely because the
nitrification-denitrification processes were developed in order to decrease ammonia-
nitrogen concentrations.
61


CHAPTER VI
CONCLUSIONS AND RECOMMENDATIONS
The BF hybrid system packed with agave fiber is suitable in the treatment of
municipal wastewater. It can remove pollutants that cause environmental problems
and effects on human health. The effluents of the BFs met the Mexican and U.S.
standards for their use in irrigation or their discharge on water bodies. The wastewater
treated within this system can be safely discharged in the environment and used for
irrigation without concern that the water is previously disinfected or applying tertiary
biological treatment. The efficiency of the agave fiber was again demonstrated as well
as its availability and economy.
The hybrid system showed the highest BOD removal efficiency at HLR of
0.54 m3 m-2 d-1 with an average concentration of 17 mg L_1 in compliance with the
NOM-003-SEMARNAT and the USA-EPA 2004 standards. The highest COD
removal efficiency was at HLR of 1.34 m3 m~2 d-1 with an average concentration of
128 mg L_1.
In FC the system showed the highest removal efficiency at HLR of 0.80 m3
m-2 d_i removing 99.99% of the concentration in the influent.13 mg L_1 was lowest
average concentration of TSS in the system and was observed at 0.80 m3 m~2 d-1. The
anaerobic filters produce effluents with low N03- concentrations (7 to 17 mg L_1)
compared with other systems. The best results were observed in the BFs with air with
exception of TSS and N3-+ Higher removal efficiency of TSS and N03- was
observed in the anaerobic BFs. BFs packed with agave fiber are excellent removers of
total suspended solids in compliance with the NOM-003-SEMARNAT-1997.
Denitrification and nitrification processes were performed. The concentration
of N03" during denitrification was low but there was a high production of N03" in the
62


aerobic stage. High concentrations of N03_ in the effluents do not solve the problem
of pollution with nitrogen components. Thus, it is suggested to change the order of the
system in order to lower the N03" production at the end of the treatment, placing first,
the aerobic BF and then the anaerobic, in order to obtain nitrification-denitrification
processes in the correct order and be able to convert all nitrogen components into gas
nitrogen.
63


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Full Text

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AGAVE FIBER BIOFILTER HYBRID SYSTEM ON THE DENITRIF ICATIONNITRIFICATION PROCESSES IN WASTEWATER TREATMENT By ADRIANA SERRANO B.S., Technological Institute of Durango, 2010 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Environmental Sciences 2014

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ii This thesis for Master of Science degree by Adriana Serrano has been approved for the Environmental Sciences Program by Casey D. Allen, Chair Azadeh Bolhari Jon Barbour July 17, 2014

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iii The result of this thesis was derived from the proj ect “Optimization of denitrification-nitrification processes with organi c biofilters treating municipal wastewater (Optimizacin de un proceso de nitrifica cin-desnitrificacin en biofiltros orgnicos tratando aguas residuales municipales, cl ave SIP20130563). It was realized in the Environmental Laboratory of Centro Interdisc iplinario de Investigacin para el Desarrollo Integral Regional Unidad Durango of Inst ituto Politcnico Nacional Unidad Durango, Mxico, directed by Professor Juan Manuel Vigueras Corts.

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iv Serrano, Adriana (Master, Environmental Sciences) Agave Fiber Biofilter Hybrid System on the Denitrif ication-Nitrification Processes in Wastewater Treatment Thesis directed by Assistant Professor Casey D. All en ABSTRACT When wastewater is discharged in the water bodies without proper treatment, it can contribute to undesirable changes in the ecosys tem as well as people. Nitrogen can be transformed into different components that might be toxic and promote the growing of aquatic plants. These components are removed by providing tertiary treatment. Biofilters (BFs) packed with organic material have demonstrated good removal of pollutants in the wastewater treatment. Thus, we ev aluated the denitrificationnitrification processes in a hybrid system of BFs w ith agave fiber as a filter media with municipal wastewater from Durango, Mexico. Two laboratory-scale biofiltration reactors were used in two trials with four hydrauli c loading rates (HLR=0.54, 0.80, 1.07, and 1.34 mm-d-), after three months of conditioning period with an HLR of 0.27 mm-d-. In this step, all BFs were fed at the top with m unicipal wastewater. Then, the BFs were connected in series. The effluen t of anaerobic BFs (BF1 and BF2) fed the aerobic BFs (BF3 and BF4) performed in dupl icate. The hybrid system showed a maximum removal efficiency of 96% in biochemical oxygen demand (BOD), 87% in chemical oxygen demand (COD), 80% in total suspe nded solids (TSS) and 4 logarithmic units in fecal coliforms (FC). Low prod uction of NO was observed in the anaerobic step (7 mg L ). High nitrification rates were observed in the a erobic BFs reaching concentrations of 222 mg L . Finally, the BF hybrid system packed with agave fiber is suitable in the treatment of municip al wastewater because it removes pollutants that cause environmental problems and it s effluents met the Mexican and

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v U.S. standards. The effluents can be safely dischar ged in the environment and reused for irrigation, prior to disinfection. In order to obtain low nitrate concentration, it is suggested to probe the hybrid system with nitrifica tion-denitrification processes. The form and content of this abstract are approved. I recommend its publication. Approved: Casey Allen

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vi DEDICATION This thesis is dedicated to my parents who have ded icated their lives to my education and well being and have supported me in e very step I want to take. I also dedicate this thesis to my loving grandparents that have always been supportive of me.

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vii ACKNOWLEDGMENT I wish to thank my committee members that were more than helpful with their expertise and precious time. A special thanks to Dr Casey Allen for his patience throughout the entire process and all the quality a dvising he was always provided. Thank you to Dr. Azadeh Bolhari and Dr. Jon Barbour for advising in the different aspects of the process and for agreeing to serve on my committee. I would like to acknowledge the CIIDIR (Interdiscip linary Research Center for Integrated Regional Development) in Durango, Mexico and specially Dr. Juan Manuel Vigueras Corts for providing all the resources and facilities for this projects development.

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viii TABLE OF CONTENTS CHAPTER I. INTRODUCTION ................................... ................................................... ..... 1 1.1 Wastewater treatments ......................... ................................................... ...... 1 1.2 Nitrogen cycle ................................ ................................................... ............ 3 1.3 Identification of the problem ................. ................................................... ..... 6 1.4 General Objective ............................. ................................................... .......... 9 1.6 Justification ................................. ................................................... ............... 9 II. LITERATURE REVIEW ............................. ................................................. 1 1 2.1 Biofilters (BFs) .............................. ................................................... .......... 11 2.2 History of filters ............................ ................................................... ........... 12 2.3 Advanced treatment ............................ ................................................... ...... 14 2.4 Bio-filters packed with organic material ...... ................................................ 14 2.5 Importance of wastewater treatment ............ ................................................ 15 2.6 Characteristics of the municipality............ ................................................... 17 2.7 Principal activities .......................... ................................................... .......... 18 2.8 Principal parameters of the wastewater ........ ................................................ 18 2.9 Established standards and guidelines .......... ................................................. 2 5 2.10 Previous studies ............................. ................................................... ......... 27 III. METHODS ...................................... ................................................... ....... 34 3.1 Hybrid system ................................. ................................................... ......... 34 3.2 Filter material ............................... ................................................... ............ 36 3.3 Wastewater influent ........................... ................................................... ....... 39

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ix 3.4 Start-up period ............................... ................................................... .......... 40 3.5 Experimental procedure ........................ ................................................... .... 40 3.6 Analytical methods ............................ ................................................... ....... 41 3.7 Experimental design ........................... ................................................... ...... 43 IV. RESULTS AND ANALYSIS ..................... ............................................... 44 4.1 Wastewater characterization ................... ................................................ 44 4.2 Organic matter (BOD and COD) removal effect .......................... .......... 44 4.3 Fecal coliform removal effect ................. ................................................ 50 4.4 Total suspended solids removal effect ......... ........................................... 53 4.5 Nitrates removal effect ....................... ................................................... 54 V. DISCUSSION .................................. ................................................... ....... 57 5.1 Organic matter (BOD and COD) removal effect .......................... .......... 57 5.2 Fecal coliform removal effect ................. ................................................ 58 5.3 Total suspended solids (TSS) removal effect ... ....................................... 59 5.7 Nitrates removal effect ................. ................................................... ...... 60 VI. CONCLUSIONS AND RECOMMENDATIONS ............... ....................... 62 REFERENCES ........................................ ................................................... .......... 64

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x LIST OF FIGURES Figure 1. Heterotrophic nitrification and aerobic denitrif ication ........................................... .. 62 Seven Nitrogen Components ....................... ................................................... ....... 223 Schematic diagrams of the two combined anaerobicaerobic BF systems............... 344 Anaerobic-aerobic BF system ..................... ................................................... ........ 355 Agave Duranguensis .................................................. ........................................... 366 Agave fiber reception ........................... ................................................... .............. 377 Agave fiber before washing ...................... ................................................... .......... 388 Preparation of the agave fiber .................. ................................................... ........... 389 Biofilter single column ......................... ................................................... .............. 3910 Wastewater collection .......................... ................................................... ............ 4011 Biofilters Columns ............................. ................................................... .............. 4112 Effluents of the start-up period ............... ................................................... .......... 4213 Effluents and influent of the last phase ....... ................................................... ...... 4214 Effluent BOD concentrations in the anaerobic BFs operated at fo ur different hydraulic loading rates. .......................... ................................................... ............... 45 15 Effluent BOD concentrations in the aerobic BFs operated at four different hydraulic loading rates. .......................... ................................................... ............... 46 16 Air*HLR interaction on biochemical oxygen demand removal in the system at four different hydraulic loading rates. ................ ................................................... ........... 48 17 COD average concentrations in the anaerobic biof ilters ....................................... 4718 COD average concentrations in the aerobic biofil ters .......................................... 4719 Air effect in the Statistical analyses of COD re moval in the hybrid system .......... 4920 HSL effect in COD removal in the hybrid system ............................................... 5021 Fecal Coliform concentrations in anaerobic biofi lters .......................................... 51

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xi 22 Fecal Coliform concentrations in aerobic biofilt ers .............................................. 5123 Air effect in the Statistical analyses of FC rem oval in the hybrid system ............. 5224 HLR effect of fecal coliform removal in the hybr id system .................................. 5325 Total suspended solids concentrations in the two biofilter series .......................... 5426 Nitrate (NO3 -) concentrations in system 2 from BF2 to BF4 ...... .......................... 5527 Air effect in the statistical analysis of NO removal in the hybrid system ........... 5628 HLR effect of NO removal in the hybrid system ..................... .......................... 56

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xii LIST OF TABLES Table 4.1 Average composition of the raw wastewater ..... .................................................. 44 4.2 BOD average concentrations in the effluents ... ................................................... 48 4.3 COD average concentrations in the effluents ... ................................................... 50 4.4 Fecal coliform removal efficiencies in logarith mic units at four different hydraulic loading rates ..................................... ................................................... ..................... 52 4.5 Total suspended solid effluent concentrations a nd removal efficiencies .............. 53 4.6 NO average concentrations in the effluents ........... ............................................ 55

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1 CHAPTER I INTRODUCTION 1.1 Wastewater treatments Raw wastewater discharged from urban centers and mu nicipalities represents one of the main contributors to water pollution. So me of the consequences of polluted water are changes in the growth rate of species, in terferences with food chains, increment of the toxicity levels, deterioration of peoples health, and impacts on ecosystem services. Water returned to the environment must satisfy many requirements. If wastewater is discharged into the hydrosphere, it m ust be treated in order to decrease toxic chemicals, pathogenic microorganisms, oxidiza ble compounds, and nutrients that support microbial growth (Ganigue, et al. 2007 ). Some examples of these components that are most of concern are ammonia, ni trates, total coliform, and infectious viruses. The principal components of the municipal wastewate r are derived from domestic and commercial sources and include; human waste, solid and dissolved forms of food waste, soaps and detergents, and soil residues. Municipal wastewater treatment plants present fluctuations in the flow a nd contaminant concentrations depending on human activities during the day (vanLo on and Duffy 2011). There are different physical, chemical, and biological treatm ents that decrease contaminant levels and allow the effluents to meet the regulati ons. The majority of these treatments occur in municipal water treatment plants. Hybrid s ystem occurs when two or more processes are combined in series (Araujo, et al. 20 08). Filtration by itself is an effective treatment that removes a large number of contaminants. The innovative technology of biofiltr ation provides a proficient

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2 treatment to the municipal wastewater. Thus, biofil ters (BFs) can treat wastewater from rural communities, agro-industry, schools, and even research centers. Organic BFs do not require a large investment during their development, have a low operating cost, are easy to operate, and take approximately o ne-fifth less space than a conventional wastewater treatment plant (Garzn-Zu iga, Tomasini-Ortiz, et al. 2008). Lately, BF systems with organic materials ha ve been used to remove organic matter, suspended material, and pathogen organisms of municipal wastewater. Activated carbon, one of the most popular materials has excellent adsorption efficiency but it is very expensive, increasing the cost of the wastewater treatment (Riahi, Mammou and Thayer 2009). Additionally, othe r types of filter materials such as peat and wood chips from different types of tree s have been used in biofiltration (Buelna and Blanger 1990; Lens, et al. 1994; Garz n-Zuiga and Buelna 2011; Gilbert, et al. 2008). Natural fibers such as cocon ut fiber, date-palm fiber, bamboo balls and agave fiber have also been used (Manoj an d Vasudevan 2012; Riahi, Mammou and Thayer 2009; Lens, et al. 1994; Vigueras -Corts, et al. 2013). These types of organic materials were selected based on t heir general characteristics, availability, use, and cost. Recently, Vigueras-Cor ts et al. (2013) obtained high removal efficiencies in wastewater treatment with i ndividual BFs using agave fiber as a packing media. Agave fiber can be obtained easily in the southeast of Durango State in Mexico. A solid waste that is inexpensive, environm entally friendly, and available in large amounts, the fiber waste is generated during mescal production (an alcoholic beverage). During mescal production, the agave is cooked, compressed, and wa shed to extract its sugars. In addition to its availabil ity in this region, agave fiber has a wide contact area that eases biofilm growth to remove wa stewater pollutants. Vigueras-

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3 Corts et al. (2013) used agave fiber as filter mat erial of a BF system where pollutant removal was developed separately in aerobic and ana erobic conditions. The system was performed using municipal wastewater from Duran go City. It decreased biochemical oxygen demand (BOD ), Total Suspended Solids (TSS), and helminthes eggs (HE) efficiently with low cost, easy operation and minimal space. On the other hand, there are standards and guidelin es for wastewater discharge that have been established by the main environmenta l agencies in Mexico and the U.S all based on physical, chemical, radiological, and microbiological properties as well as in specific elements (vanLoon and Duffy 2011). W ater quality guidelines depend on the final use of the water. Usually, water used for human consumption requires strict regulations whereas water reused for agricul tural or industrial goals has less strict regulations that do not account for removal of toxic chemicals and harmful microorganisms. 1.2 Nitrogen cycle Nitrogen, along with water, carbon, and phosphorus has its own cycle (Withgott and Brennan, 2011). Nitrogen cycle has di fferent processes such as, nitrogen fixation, nitrification, anammox, and deni trification that transform nitrogen into different forms. During nitrogen fixation, gas nitrogen is reduced to ammonia, a biologically available nitrogen form. The biologica l reduction is catalyzed by nitrogenase, a multimeric enzyme complex (Berman-Fr anka, Lundgren and Falkowski 2003). Thus, atmospheric nitrogen enters into the c ycle mainly by nitrogen-fixation, where it is converted to organic nitrogen (Latyshev a, et al. 2012). Nitrification and denitrification are processes tha t occur naturally in the environment, they occur in water, in soil, and in t he atmosphere (M. S. Jetten 2008). Because they remove undesirable nitrogen components nitrification and

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4 denitrification processes have been simulated in pr evious research (Rebah, et al. 2010; Li, et al. 2013). Simulation of nitrification and d enitrification processes can be performed providing aerobic and anaerobic condition s. Thus, nitrogen components that are dangerous to human health and the environm ent can be oxidized in simulated scenarios. In the nitrification process, ammonia is oxidized b y the autotrophic nitrifying bacteria in two steps. First, ammonia is oxidized t o nitrite by ammonia-oxidizing bacteria (AOB) in aerobic conditions and then nitri te is oxidized to nitrate by nitriteoxidizing bacteria (NOB) (Zhang, Love and Marc 2009 ). Nitrosomonas convert ammonia under aerobic conditions to nitrite derivin g energy from the oxidation (Equation 1). Nitrite is oxidized by Nitrobacter to nitrate (Equation 2) (Posmanik, Gross and Nejidat 2014): nn n (1) n nn (2) The nitrate formed can be used positively as a nutr ient for the plants. Nevertheless, when there is an excess, it percolate s into the water flowing through the soil because it cannot retain the overabundance amo unts of nitrate (Yoshimoto, et al. 2013). Thus, groundwater reservoirs can enclose hig h concentrations of nitrates which cause extensive environmental problems. During this stage, nitrogen forms change but are not assimilated. Nitrification is important for three main reasons: 1) nitrification represents a vital phase in the nitrogen cycle beca use of the production of nitrate, 2) if nitrite is not oxidized, it can negatively affect p eoples health, and 3) nitrifying bacteria are usually competing with primary produce rs, so due to nitrification,

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5 nitrifying bacteria are present only when ammonium is present in high concentrations (Dodds 2002). Denitrification is the other part of the nitrogen c ycle. It develops the biological reduction of nitrate to nitrogen gas (N ) by facultative bacteria. This process is developed under anaerobic conditions where the bact eria use nitrate as the primary source of oxygen (Busigny, et al. 2013). The anoxic conditions for denitrification require a dissolved oxygen concentration of maximum 0.5 mg/L. When the bacteria break down nitrate in order to take the O nitrate is reduced to nitrous oxide (N 0) and then it turns to nitrogen gas (Equation 3) (Mat eju, et al. 1992) NO NO NO + N O N (3) Nitrogen gas (N ) has low water solubility. Thus, it escapes to the atmosphere as gas bubbles (Harter, et al. 2014). Since N is the principal component of air, small releases do not cause environmental concern. During denitrification, a carbon source is required and in wastewater treatments, the carbon source is obtained from the wastewater. Organic carbon is oxidized to CO and cellular energy (Equation 4), with water and h ydroxyl ions as end products (Mateju, et al. 1992) 6NO + 5CH OH 3N + 5CO + 7H O + 6OH (4) Nitrification takes place under aerobic conditions, however, according to Wen and Wei (2011) anaerobic nitrification of ammonia c an be performed by heterotrophic

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6 bacteria that use the organic matter (reductases) o f the wastewater as the carbon source. Denitrification process under aerobic conditions wa s demonstrated by Wen and Wei (2011), Shi, et al. (2013), Chen and Ni (20 12), and Joo, Hirai and Shoda (2005), also shown in figure 1. Reductases: Ammonia monooxygenase (AMO), hydroxyla mine oxidase (HAO), periplasmic nitrate reductases (NAP), nitrite reductases (NIR), nitric oxide reductase (NOR) and nitrous oxide reductase (NOS) (Wen and Wei 2011). Figure 1 Heterotrophic nitrification and aerobic denitrifica tion Nitrification and denitrification processes occur n aturally in the environment in such mediums as water, in soil, and in the atmos phere (M. S. Jetten 2008). Because they remove undesirable nitrogen components, nitrif ication and denitrification processes have been simulated in previous research (Rebah, et al. 2010) (Li, et al. 2013). Simulation of nitrification and denitrificat ion processes can be performed providing aerobic and anaerobic conditions. Thus, n itrogen components that are dangerous to human health and the environment can b e oxidized in simulated scenarios. 1.3 Identification of the problem The development and improvement of methods for biol ogical wastewater treatment are very important, particularly methods that remove nitrogenous contaminants. High concentrations of some nitrogen forms can cause significant

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7 atmospheric, terrestrial, and aquatic problems (Har ter, et al. 2014). The removal of nitrogen components in wastewater represents a tert iary treatment but it is essential to reduce their concentration in the effluents to disc harge in receiving bodies. During degradation of organic matter, an aerobic BF by itself produces different nitrogen-nitrate (N-NO3 -) components. Ammonia-nitrogen and nitrates have serious effects on the environment and human health In human health, ammonia irritates tissues and alters the uptake of oxygen b y hemoglobin decreasing oxygenation of tissues (Holeton, Chambers and Grace 2011). High levels of nitrate in waters are also considered a threat to the public h ealth because they can be ingested by eating vegetables or drinking water. Drinking wa ter containing large amounts of nitrate causes methemoglobinemia in infants in 1962 (Vigil, et al. 1965). Methemoglobinemia is derived from the interaction b etween nitrite and hemoglobin where nitrate converts to methaemoglobin-producing nitrite. This causes hypoxaemia due to an oxygen dissociation curve (Chan 2011). Hi gh nitrates levels also increase cancer risk due to reduction of nitrates to nitrite s that can later form N-nitroso compounds (NOCs). NOCs are dangerous carcinogens an d act systematically (Njeze, Dilibe and Ilo 2014). In the environment, the presence of ammonium and ni trates-nitrogen in large amounts in reservoirs, subsoil, and groundwater can cause catastrophic deterioration of fresh water quality (Guerra, et al. 2013). All n atural bodies of water are able to oxidize organic matter when the nitrogen loading -p rincipally ammoniais maintained within the limits of the oxygen resources. When the nitrogen loading exceeds the oxidative capacity of the water bodies, degradation of nitrogen forms is inhibited. Acceptable levels of dissolved oxygen, calculated t hrough research, also determine if forms of aquatic organisms can survive (Rezaei, et al. 2013).

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8 In the past, oxygen sources were the pollutants wit h higher concern in surface waters, but nowadays, pollution of surface and grou ndwater with contaminants from industrial and agricultural sources is the highest concern. During the last 50 years, a variety of chemical have been developed for agricul tural purposes, such as fertilizers and pesticides, that are principally composed of ni trogen and phosphorous components (William, Brjesson and Hedlund 2013). N itrogen forms are essential fertilizers to the algae growth. Algae growth is no t really environmentally desirable and it can occur when water with nitrogen forms are discharged into bodies of water (Li, et al. 2013). If wastewater with ammonia and n itrate-nitrogen is discharged into lakes, streams, or wetlands, it can cause eutrophic ation “the process of an ecosystem becoming more productive by nutrient enrichment sti mulating primary producers” (Dodds 2002). Nutrients such as inorganic nitrogen promote the growth of undesirable aquatic plants and algae. Algal blooms growth is on e of the worst effects of Eutrophication. They cause undesirable taste, odor, and color issues and increase when lakes become more eutrophied (Martinez-Lopez, et al. 2007). The oxidation of rivers and estuaries is another pr oblem caused by dissolved inorganic nitrogen forms. During the autotrophic co nversion of ammonia to nitrate, oxygen is required. Thus, when ammonia is discharge d into rivers and estuaries, its oxidation can cause a reduction of dissolved oxygen levels (Bresler 2012) which can be even worse when long residence time for the grow th of nitrifying bacteria is available. Ammonium nitrogen exists as un-ionized ammonia (NH ) or ionized ammonia (NH ). Un-ionized ammonia is more toxic than NH because it is uncharged and lipid soluble (Gao, et al. 2011). In addition, toxi city is also determined by pH levels. Concentrations above 0.2 mg/L of free ammonia can c ause death in many species of

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9 fish and the National Research Council Committee in the U.S. recommends this amount as the limit permitted in receiving bodies ( Sawyer, McCarty and Parkin 2003). Thus, wastewater should be treated in order to decr ease concentrations of inorganic nitrogen forms that may damage the enviro nment and human health. 1.4 General Objective The main objective of this study rests in evaluatin g the denitrification and nitrification processes in a hybrid system of biofi lters using agave fiber as filter media with four different superficial hydraulic loads wit h the intent of meeting Mexican and US EPA effluent regulations. This research intends to specifically improve the k nowledge and application of BFs by: Determining the removal efficiencies of organic mat ter, suspended material, nitrates and ammonia-nitrogen in the wast ewater treatment when anaerobic and aerobic conditions are provided and Determining the maximum hydraulic loading when the effluents meet the regulations of the municipal wastewater treatme nt and the effluents can be reused in irrigation crops and green areas. 1.6 Justification The organic BFs will contribute to decrease polluti on and environmental problems of receiving bodies by the discharge of tr eated effluents without nitrogen components. Discharging effluents where nitrogen co mpounds have been removed will decrease problems such as eutrophication, oxid ation, and high levels of toxicity in rivers, lakes, and estuaries (Cotman, Zagorc-Kon can and Drolc 2001). Hybrid

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10 biofiltration systems are inexpensive and sustainab le method for this process that can be applied to decrease the impacts of wastewater di scharges. The principal goal of removing nitrogen compounds from wastewater is the protection of the environment. In addition to the discharge of water without pollu tion, the water treated in this system can be reused in productive activities such as industry and agriculture, in places with lack of treatment plants and scarcity o f water. Regulations and quality guidelines are less strict for industrial purposes and irrigation. They are based on the concentration of soluble salts, and potentially tox ic elements as well as the molar ratio of sodium to calcium and magnesium (USA-EPA 2012). All these chemical components are in very low quantities or not contai ned in effluents. In order to prove the low concentrations of these chemicals, conducti vity and alkalinity analyses were developed. Aesthetics criteria are generally unnece ssary, however, odor and color are very suitable in the wastewater treated by the orga nic BFs.

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11 CHAPTER II LITERATURE REVIEW This section outlines specific and relevant literat ure related to this research agenda. First, I defined BFs and their main categor y followed by the historical development of filtration in wastewater treatment. Then, after discussing a vast history of filters I briefly discuss advanced treat ments and introduce the first BFs packed with organic material. Subsequently, the imp ortance of wastewater treatment is described to continue with the characteristics a nd principal activities of Durango municipality that provides a general idea of the po ssible wastewater constituents in this area. The principal parameters in the wastewat er are described as well as the established standards and guidelines of the environ mental agencies. Finally, previous studies using organic BFs or/and aerobic and anaero bic conditions and achieving nitrification and denitrification are presented. 2.1 Biofilters (BFs) The trickling filter (TF) is a “fixed biological be d of rock or plastic media on which wastewater is applied for aerobic biological treatment” (Wang, et al. 2009). These attached growth systems use a media of granit e, limestone, clinkers, plastic tubs, or hard coal that distribute and contact the wastewater. A biological slime layer called biofilm is grown in the filter media. The re moval of the dissolved organic pollutants takes place due to their biological oxid ation developed by the slime film and their followed degradation by the aerobic and f acultative bacteria. The biological filter (BF) is a type of trickling filter that is a ble to operate aerobically and anaerobically. The biofilm contains microorganisms, particulate material, and extracellular polymers. In addition to the inorgani c media that trickling filters use, organic media such as fibers, compost and wood are also used to pack biofilters.

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12 Organic packing material is a good supporting mater ial that allows the attachment of microorganisms (Rebah, et al. 2010). Degradation of organic matter and other pollutants and retention of suspended solids have b een successfully developed in these systems. 2.2 History of filters The trickling filter was installed for the first ti me at the Lawrence Experiment Station in Massachusetts in 1891. It had a distribu tion by spray nozzles (Wang, et al. 2009). However, it was during the 20th century that the majority of improvements in wastewater management were developed. In 1940, biol ogical filtration had a great impact and most of the wastewater treatment plants in the United States had trickling filtration. In 1946 the National Research Council d eveloped a mathematic formulation for the trickling filter design (Daiggerl and Boltz 2011). In addition, the membrane bioreactor (MBR) which was one of the most importan t tools in the treatment of waste liquids and solids was introduced in the mid-1960s Some of the advantages of MBR are; high removal of pathogens and contaminants, mi nimal footprint, few energy demand, low cost, and easy operation (EPA 2007). Th e MBR treatments include two steps; bacterial degradation of organic matter in t he presence of dissolved oxygen developed in a bioreactor, and the separation of su spended solids and bacteria from the effluent developed by the membrane in a second step (Sutherland 2010). These processes make up the complete treatment -primary, secondary and even part of the tertiary treatment-that is developed in a conventio nal wastewater treatment plant. Dorr Oliver introduced the idea of combined sludge digestion into a very fine filter with flat plate membranes in a side-stream l oop in the mid-1960s. In this decade, microfiltration and ultrafiltration membranes were developed at a commercial scale (Baker 2000). However, the emerging technology was very expensive. It wasnÂ’t until

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13 1989 in Japan that the MBR started to be widely use d due to the initiative of the Japanese government to find efficient and low cost methods to wastewater treatment. This new version emerged in the European market in the mid-1990s. After the 1990Â’s the MBR field experienced extensive growth. Many re searchers started developing new technologies in this field. Today there are mor e than 3000 plants around the world that use this technology (Stephenson, et al. 2000). While MBR was expanding, the trickling filter exper ienced some improvements. In the early 1960Â’s, Imperial Chemica l Industries, Ltd. initiated the use of plastic media in TF. Plastic media allows th e growth of the biofilm in its area. Plastic materials were a success because of the hig h-rate trickling filter media that was obtained (Wang, et al. 2009). The anaerobic biofilter was another improvement to the attached growth wastewater treatment. It was studied in 1962, for t he first time by James Young and Perry McCarty (Kaiser, Dague and Harris 1995). The anaerobic biofilter was packed with different types of filter media. No oxygen was provided to this system, which decreases energy use. During this biofiltration, an anaerobic carbonaceous oxidation of the organic matter is performed. Anaerobic biofi ltration was used to remove nitrogen components because it provides suitable co nditions to achieve denitrification. However, improvements in the nitrification process are still needed. High carbonaceous removal at low cost and low sludge pro duction is achieved during anaerobic biofiltration. The biofilter performs the same process as MBR but it does it in one-step. The packing material allows the growth of a biofilm and the suspension of organic matter. Thus, the degradation of pollutants is developed by the attached microorganisms and the suspended solids are trapped in the packing mat erial.

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14 2.3 Advanced treatment Due to the understanding of the impact of wastewate r and their pollutants such as phosphorous and nitrogen compounds, advanced tre atments to these pollutants were developed. Once the reduction of carbonaceous and other pollutants was achieved through the development of secondary treat ment, the prevention of eutrophication by the phosphorous and nitrogen comp ounds was the next step in the history of wastewater treatment. The firsts in deve loping the denitrification process to the wastewater treatment were Ludzack and Ettinger (1962). They used an anoxic zone to achieve biological denitrification in an ac tivated sludge process. Barnard (1973) developed and patented a single sludge syste m that biologically removed nitrogen and phosphorous (Lofrano and Brown 2010). 2.4 Bio-filters packed with organic material In 1987, the use of organic media in biofiltration was studied. Rana and Viraraghavan (1987) developed a biofilter packed wi th peat in The U.S. and Canada that showed a good removal of pollutants, even nitr ogen. During the second half of the 1990s and the beginning of the 2000Â’s biofiltr ation through organic media was widely performed. Organic BFs were applied in rural and semi-urban regions where there were no wastewater treatment plants and the w astewater could be treated in situ. The organic materials used in biofiltration were co mmonly endemic of the area where the biofilters were developed. For example Garzn-Z uiga, Tomasini-Ortiz, et al. 2008 developed a trickling biofiltration in Morelos Mexico using sugar cane fibers which are very common in this area. Riahi, Mammou a nd Thayer 2009 developed a BF in Tunisia packed with Phoenix dactylifera which stretches from North Africa to the Middle East. There are many other examples of o rganic BF that are described below.

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15 2.5 Importance of wastewater treatment Wastewater from industry and domestic use should me et a specific set of requirements before being returned to the natural e nvironment. The levels of toxic chemicals and microorganisms contained in wastewate r depend on the source because the ecosystem is not able to transform high levels of pollutants. In some cases, municipal wastewater that contains domestic and ind ustrial wastes is not treated before to discharge into water bodies such as river s, lakes, and oceans. The typical components of municipal wastewater are human waste, food waste, soaps, detergents, and soil residues. The level of pollution in wastew ater is determined by measuring parameters such as biochemical oxygen demand (BOD ), chemical oxygen demand (COD), total suspended solids (TSS), Total phosphor ous (TP), and total nitrogen (TN). Thus, when wastewater is discharged into the hydrosphere it should not contain high levels of toxic chemicals dangerous to the eco system, oxidizable components, nutrients that can cause microbial growth or pathog ens (vanLoon and Duffy 2011). Levels of BOD TSS, TP, and TN are the parameters with the great est concern. These parameters indicate the concentration of pollutants that affect the environment and interfere with the marine life. When wastewater is going to be used for irrigation, it is not required to remove nutrients or benign dissolved and suspended organic matter. However, toxic chemicals and microorganisms represent undesirable components and should be removed (U.S. Environmental Protection Agency 2012) When wastewater will be discharged in to the hydrosphere, in addition to to xic chemicals and microorganisms, nutrients and dissolved organic matter need to be r emoved. Water bodies have sufficient levels of dissolved oxygen to oxidize an imal and vegetative wastes through aerobic microbial reactions. During this process, d issolved oxygen is converted to

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16 CO (vanLoon and Duffy 2011). Carbon dioxide is then us ed during the photosynthesis and converted again to oxygen. This natural cycle keeps the natural ecosystems in a stable state. When excessive amount s of dissolved oxygen are provided by wastewater discharge, the self-purifica tion cycle is broken (Sonune and Ghate 2004). Excessive amounts of degradable organi c matter produce anoxic conditions which reduces important aerobic biologic al degradations. The TSS of the untreated wastewater cause an increase of turbidity which inhibits photosynthesis. Nutrients such as phosphorous, nitrogen, and silica te are important elements to the aquatic systems. Most of the plants use them fo r growth and reproduction (Biszel and Uslu 2000). However, the enrichment of nitroge n and phosphorous has negative effects on aquatic ecosystems. High levels of nitro gen and phosphorous stimulate plant and algal growth. Inorganic nitrogen and phos phorous become available for phytoplankton production in aquatic systems heavily contributing to eutrophication. Nitrogen can be present in wastewater discharges th rough different inorganic nitrogen forms such as ammonia nitrogen, nitrates, and nitri tes. As it was mentioned before, dissolved inorganic nitrogen forms can cause oxidat ion of rivers and death in many species of fish. In order to protect aquifers, regulations have been established by the government where permissible levels of contaminants are stated. Considering the final use, the effluents of treatment facilities are requ ired to meet specific standards. The established standards and guidelines are described later. Because of these guidelines, a variety of wastewater treatments processes have b een developed in order to clean water and achieve effluent regulations. The typical treatment includes a primary and secondary treatment where suspended particles are r emoved and organic matter is converted to bacterial biomass, H O and CO Some processes include a tertiary

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17 treatment where disinfection is performed or specif ic contaminants such as nitrogen and phosphorous are removed (vanLoon and Duffy 2011 ). These treatments include activated sludge process, chemical coagulants for t urbidity removal, sludge digestion, and attached growth systems. They may include physi cal, chemical, and biological processes. Some of them have been established in bi g scales treating up to 10,000 m of municipal wastewater every day (Tchobanoglous, B urton and Stensel 2004). All these processes and designs are frequently improved or replaced. 2.6 Characteristics of the municipality The municipality of Durango is located in the Valle del Guadiana in the north of Mexico. The word Guadiana means “Wide River” in the Arabic etymology. Due to The Tunal River located in the Valle del Guadiana, this name was assigned to the area since the first human settlement during the Colonia l era. Durango City is the most important city in the municipality of Durango and i s also the capital of the state that bares the same name. Durango is located at 24.2 no rth latitude and 104.4 west longitude with an elevation of 1,880 m above sea le vel. The climate in Durango is semi-arid with low precipitation during the summer and semi-cold during the winter. The average temperature is 17.5C (INEGI 2013). Durangos geology comprises the Cenozoic (70.9%), t he Quaternary (23.6%), the Neogene (4.1%), and the Paleogene (0.1%) period s. Recent alluvial deposits, basaltic rocks, and extrusive igneous rocks can be found in the area (INEGI 2013). Durango is the city with the biggest population in the state of Durango. At the end of the eighteenth century, -three centuries after the official foundation of Durangothe city had a population of 7400 inhabitants in 1500 h ouses. The streets where traversed by canals that carried water from the wells to dome stic use and irrigation. During the 1900’s water was provided to the population through pressure pipes. It wasn’t until

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18 1969 when the potable water and sewage systems were introduced to some places of Durango City. In 1969 the potable water and sewage systems had 57,370 m and 95,915 m of pipe network respectively. In 1987 the pipe network of sewage was extended to 175,494 linear meters and 3,400 hookups where added to the 12,500 that previously existed. During this period, 6 oxidation ponds to wastewater treatment were built on an 86 hectares plot of land (Aguas de l Municipio de Durango 2013). The oxidation ponds were located where the municipa l wastewater treatment plant is currently situated. The wastewater used in this pro ject was collected from the municipal wastewater treatment plant in Durango Cit y. 2.7 Principal activities The sewage system indirectly provides information o f the characteristics – customs and behaviorsof the population from food to hygiene habits as well as from the use of pharmaceuticals and birth control pills (Lofrano and Brown 2010). The majority of the municipal wastewater from Durango c omes from domestic and commercial use. Industrial use is also included in the sources of municipal wastewater but it does that in few quantities. VanLoon and Duffy 2011 affirm that the highest leve l of flow and contaminant concentration in the sewage are found d uring the morning and evening. In the specific case of Durango, Mexico, due to the population’s activities, higher values of contaminant concentration of the municipa l wastewater treatment plant are found at 2:00 pm and 6:00 pm (Vigueras-Corts, et a l. 2013). 2.8 Principal parameters of the wastewater When developing wastewater treatments reducing the occurrence of specific parameters remains a key goal. These parameters inc lude: biochemical oxygen demand, chemical oxygen demand, total solids, total nitrogen, and fecal coliform.

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19 2.8.1 Biochemical Oxygen Demand (BOD ) BOD is the amount of dissolved oxygen required by b acteria to degrade decomposable organic matter in aerobic conditions ( Sawyer, McCarty and Parkin 2003). This parameter indicates the organic quality of the water by the determination of biodegradable organic compounds (Riedel, et al. 1988). The BOD test indicates the oxygen that is required by the aquatic ecosystem to oxidize the organic matter once it is discharged. If the BOD level is high, it inhibit s the purification capacity of the water systems. Thus, BOD data should be interpreted in terms of organic matter and as the amount of oxygen used during its oxidation. The oxidation reactions are governed by the number of microorganisms as well as the temperature. The BOD test is performed in a constant temperature of 20C that is a usual value in natural bodies of water. The complete organic matter oxidation the oretically takes an infinite amount of time. However, for practical purposes, 20 days m ay be enough for complete oxidation. Although 20 days sounds more practical, it remains a substantial period of time. After many experiments, it has been found tha t in 5 days a large percentage of the total BOD is exerted. Thus, a BOD test with a 5 -day incubation period is the current basis for determination of this parameter ( Sawyer, McCarty and Parkin 2003). This method is called the BOD It is important to keep in mind that only a porti on (70 to 80 percent) of BOD is determined by this method due to the length of the period of time that it is performed in. Another advantage of the BOD is that during the 5-day incubation period it minimizes the possibility of o xidation of ammonia. The typical BOD in municipal wastewaters is between 100-300 mg L and depends on previous water use (vanLoon and Duffy 2011). The goal of BOD in a wastewater treatment is below 30mg L which is the permissible level of re gulations (“The Clean Water Act” and “NOM-003-SEMARNAT-1997”).

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20 2.8.2 Chemical Oxygen Demand (COD) The COD is measured in order to determine the organ ic strength of domestic and industrial wastes. It represents the total quan tity of oxygen required for oxidation of organic matter in order to obtain carbon dioxide and water. This oxidation is known to be performed in all organic compounds by t he action of strong oxidizing agents under acid conditions. During COD determinat ion, all the organic substances are oxidized even if they are not able to assimilat e biologically. The amount of oxygen used during the oxidation process is related to the amount of organic matter contained in the wastewater. Thus, COD values are h igher than BOD values. When wastewater contains high amounts of biologically re sistant organic matter, COD values are greater because of the amount of oxygen required for the oxidation to be high. Glucose and lignin are examples of organic su bstances that require large amounts of oxygen to oxidize. Measuring BOD provides more advantages than measuri ng COD. The COD test is less suitable because of the limitation dif ferentiating biologically oxidizable and inert organic matter. COD results provide an es timate of the amount of all organic matter types together. COD test does not provide a real rate of organic matter oxidation as it occurs in nature. However, COD requ ires less time (3 hours) to measure than BOD. COD values can be interpreted in terms of BOD values by establishing a reliable correlation between COD and BOD. The typical COD in municipal wastewaters is 500 mg L- and it depends on previous water use (vanLoon and Duffy 2011). 2.8.3 Total Solids Solid matter is all of the matter contained in a li quid material except for the water. However, solids are defined as the matter th at remains as residue upon

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21 evaporation and drying at 103-105C (Mines Jr. 2014 ). Solid tests are empirical in character and very simple to perform due to the wid e variety of inorganic and organic materials encountered. Total suspended solids are t he particles of more than 2.0m of diameter that are retained by the filter. Total dis solved solids are found in potable water in the form of inorganic salts, small amounts of organic matter, and dissolved gases. The hardness of the water increases with tot al dissolved solids. Total suspended solids (suspended colloidal and larger matter) incr ease with the degree of pollution. Determination of total suspended and total dissolve d solids is developed by the measurement of filtered and unfiltered portions of samples (Sawyer, McCarty and Parkin 2003). Suspended solids cause aesthetic issues, a decline in aquatic species, and ecological degradation of aquatic environment. Some of the effects of suspended solids are the reduction of the amount of light pen etrating into water which reduces availability of energy for species survival (Bilott a and Brazier 2008). The typical content of total solids in municipal wastewaters is approximately 720 mg L. Total suspended solids are between 100-350 mg L (vanLoo n and Duffy 2011). The goal of total suspended solids in wastewater treatments which is also defined by regulations is below 30mg L (“The Clean Water Act ” and “NOM-003SEMARNAT-1997”). 2.8.4 Total Nitrogen Nitrogen components are contaminants of great conce rn because of the effects that they have on the environment as well as on hum an health. Due to the extensive use of fertilizers and pesticides, nitrogen compone nts are found in wastewater in high quantities. Due to the variety of oxidation states that nitrogen presents, there are many nitrogen components. There are a total of seven oxi dation states that result in the

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22 formation of seven components, all of them are very important to the environment; ammonia, nitrogen gas, dinitrogen monoxide, nitroge n monoxide, dinitrogen trioxide, nitrogen dioxide, and dinitrogen pentoxide (Figure 2) (Doyle and Hoekstra 1981). Figure 2 Seven Nitrogen Components NH N O and N O mixed with water form inorganic ionized species; ammonium, nitrite, and nitrate (Equations 5, 6 and 7), which are of environmental concern in water and they are regulated (Bresler 20 12). NH + H O NH + OH (5) N O + H O 2H + 2NO (6) N O + H O 2H + 2NO (7) All nitrogen components can cause environmental pro blems except for N which is the main component of the atmosphere. The four forms of nitrogen that are of interest in water resources are ammonia, nitrite nitrate, and organic nitrogen.

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23 Ammonia nitrogen represents all nitrogen that exist s as an ammonium ion or as ammonia. The nitrogen contained in organic compo unds is the organic nitrogen. Amino acids, nucleic acids, amines, amides, imides, and nitro derivatives are examples of organic nitrogen components. Organic ni trogen components have little significance in water analysis. Nitrates and nitrites are nitrogen-oxygen chemical units mixed with organic and inorganic compounds. Colorimetric procedures an d ion chromatography are used to determine nitrites due to the sensitivity requir ed to measure them. Nitrate levels are difficult to determine. Some of the methods used to determine nitrates are: ultraviolet spectrometry, chromatographic and capillary ion ele ctrophoresis, nitrate electrode, and cadmium reduction (Senra-Ferreiro, et al. 2010) Total nitrogen (TN) is the sum of ammonia nitrogen, organic nitrogen, nitrates, and nitrites. Gas nitrogen is not conside red part of TN because of the nonreactive effect that it has in water. TN gives a general idea of the nitrogen component levels in wastewater. Nitrogen components remaining in polluted waters ar e Indicators of Sanitary Quality. When water contains mostly nitrate it mean s that water was polluted a long time ago. This assumption is based on the oxidation sequence of the nitrogen components. Typically, polluted waters contain orga nic nitrogen as the primary pollutant. After some time, organic nitrogen is con verted to ammonia nitrogen. If aerobic conditions are present ammonia will be oxid ized to nitrite and nitrate (Rssle and Pretorius 2001). The typical content of TN in m unicipal wastewaters is approximately 30-40 mg L (Taylor 2013). Nitrates ar e not usually present in Municipal wastewater (Ahmed, et al. 2012).

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24 2.8.5 Fecal Coliform Fecal coliform bacteria are microorganisms found in feces. They are facultative anaerobes –they can survive in the abse nce of oxygen-, gram-negative, non-spore forming, and they ferment lactose produci ng gas and acid when incubated at 35C (Department of Environmental Sciences 2003) Although fecal coliforms are usually not disease-causing organisms, they are tes ted to identify more harmful present bacteria in the wastewater (Francy, et al. 2004). Fecal coliform are indicators of fecal pollution and of the presence of enteric p athogens of wastewater (Mack 1977). The test provides an easy and fast way to me asure microorganism concentration avoiding the use of additional tests to determine enteric viruses that are difficult and time consuming. Fecal coliform is a g ood technique to determine fecal contamination. It is also good as an indicator of p athogen regrowth because of the fact that viruses and parasites cannot reproduce without a warm-blooded host (Efstratiu, et al. 1988). However, the absence of fecal coliform d oes not mean that fecal contamination is absent. Fecal coliform is also les s reliable as an indicator of viruses and parasites. Another disadvantage of fecal colifo rm is that during wastewater treatment, some pathogens are more resistant than f ecal coliform and they are not readily removed. For example, viral pathogens have a greater survivability than fecal coliform during anaerobic digestion. Thus, the meas urement of fecal coliform after a treatment may not provide accurate determination of all the pathogens (Department of Environmental Sciences 2003). On the other hand, fe cal coliform is a good indicator of Salmonella sp Fecal coliforms are the best predictors of the pr esence of Salmonella sp (Efstratiu, et al. 1988). There are different methods to measure fecal colifo rm. One of the most popular is the multiple-tube fermentation (MTF) whi ch is performed using different

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25 dilutions (Gronewold and Wolpert 2008). Fecal colif orms are identified by gas production present in the Durham tubes. The average concentration of fecal coliform in municipal wastewater is between 10 and 10 n cells L . The FC concentration should remain below 1.0E 03100 mL according to Garzn-Zuiga and Buelna (2011). 2.9 Established standards and guidelines Wastewater treatment and guidelines were not genera lly taken into consideration for centuries. Wastewater was dispose d of or discharged without any treatment or regulation. Because of this, serious i mpacts on public health and the environment were caused. The emergence of epidemics in Europe during the nineteenth century was one of the critical effects of the incorrect disposal of the wastes. In order to protect the health of the popul ation and the environment, scientific discoveries, debates on societal priorities, and go vernment interest allowed the establishment of standards and guidelines for the d isposal and management of the wastewater. The first “Water pollution control” reg ulation in the U.S. was put into effect in the British colony of Massachusetts in 16 47 (Lofrano and Brown 2010). The major revolution in wastewater treatments and their regulation took place during the 20th century. The concepts of Biochemical Oxygen Deman d (BOD ) and other standards were presented in the Eighth Report (1912 ) of the Royal Commission on Sewage Disposal. These standards were also applied in many other countries and they began to mandate wastewater treatment. In 1950 afte r some debates of water quality standards and stream use classification, the develo pment of waste management policies began. In 1948 the US government, through Congress, enacte d the first Federal Water Pollution Control Act. The law did not provide enou gh regulations to control water

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26 pollution and gave limited authority to the federal government. The law was then continuously amended until 1972 when an appropriate legislation was obtained. The Federal Water Pollution Control Act or Clean Water Act focuses on eliminating or reducing the pollution of interstate waters and tri butaries and improving the sanitary condition of surface and underground waters. Nowadays, the Environmental Protection Agency (EPA) is the primary authority for the implementation of the Clean Water Act in the U.S. The EPA sets effluent limits that apply to different water disch arges to ensure the protection of the receiving water bodies. The maximum national standa rds for secondary treatment in the U.S. are 30 mg L in BOD 30 mg L in TSS and a range between 6 and 9 in pH on an average 30-day concentration (Tchobanoglous, Burton and Stensel 2004). For total nitrogen, the U.S. Environmental Protection A gency established a total nitrogen range from 0.12 to 2.18 mg L depending on the sensitivity of the region (Galil Malachi and Sheindorf 2009). In Mexico, SEMARNAT (Secretary of Environment and N atural Resources) is the principal authority determining wastewater para meters by the implementation of the official regulation NOM-003-SEMARNAT-1997. Ther e the maximum permissible limits of pollutants in treated wastewa ter are 30 mg L in BOD 30 mg L in TSS, 1.00E + 03100 ml in FC and a range betwe en 6 and 9 in pH (SEMARNAT 1997). For total nitrogen the NOM-001-SEM ARNAT-1996 established 15 mg L as the maximum permissible limit for the aquatic wildlife protection.

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27 2.10 Previous studies 2.10.1 Organic biofilters Wastewater treatments using bio-filters packed with organic materials have shown efficiency in pollutant removal. Some of the biological residuals used for packing material, also called filter media, are aga ve, compost, peat, soil, wood shell, wood chips, heather, as well as coconut, date-palm, and bamboo fibers (ViguerasCorts, et al. 2013; Lens, et al. 1994; Buelna and Blanger 1990; Garzn-Zuiga and Buelna 2011; Gao, et al. 2011; Nicolai and Janni 20 01; Riahi, Mammou and Thayer 2009). The biofilter should have a porous solid med ia that allows the support of microorganisms and the access of pollutants in the airflow (Nicolai and Janni 2001). Thus, pollutants are removed through degradation wi th microorganisms. Biological residuals such as date-palm fibers are natural, ine xpensive, and environmental friendly materials that can be used toward biofiltr ation (Riahi, Mammou and Thayer 2009). There have been many studies that used different ty pes of packing materials. Riahi, Mammou and Thayer performed an experiment us ing columns packed with Phoenix dactylifera one of the most cultivated palms around the world. Date-palm fibers were chosen because of their wide waste avai lability as well as large amounts of waste after the trimming operations. Garzn-Zui ga and Buelna (2011) developed a trickling biofilter (TBF) that was packed with a blend of pruning waste from two tree species; Dwarf Poinciana (Caesalpina pulcherrima) and Jacaranda (Jacaranda mimosifolia) which are ornamental trees obtaining removal effici encies of 97% in BOD 71% in COD, 95% in TSS and 4 log units in fecal c oliforms. Organic biofilters are an efficient and economical method to decrease turbidity, phosphorous, chemical oxygen demand (COD ), and helmith eggs of

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28 secondary domestic water. Organic media provides ad equate pollutant removal because it allows the microorganismÂ’s growth. Thus, biomass accumulation is possible inside of the reactors and the biodegradat ion of the pollutants is improved. There are also some experiments that did not use or ganic material at all. Rebah et al. (2010) developed two different biofiltration system s using clay granular media in one of the systems and plastic media in the other. Reba h developed aerobic and anaerobic scenarios in each system in order to provide the ne eded conditions to the nitrification and denitrification processes. Recently, Vigueras-C orts et al. (2013) developed two series of bio-filters using agave fiber in the muni cipal wastewater treatment process. Agave fiber waste is generated during mescal produc tion. Agave fiber is a solid waste inexpensive and environmentally friendl y. It also has a wide contact area which facilitates biofilm formation that degrades w astewater pollutants. They evaluated laboratory scale biofiltration reactors i n two trials, installing aerobic and anaerobic bioreactors. They used five different hyd raulic loading rates (HLRs) of 0.27, 0.54, 0.80, 1.07, 1.34 m m d obtaining removal efficiencies of 92% in BOD 79% in COD, 99.9% in fecal coliforms and 91% in T SS. BFs have been developed using different column mate rials and different dimensions. For example, Rebah et al. (2010) used P lexiglass columns of 2-1.4 m by 0.2 m. Riahi et al. (2009) set up three pilot scale reactors with a 0.55 m long glass column and 0.06 m of internal diameter. Vigueras-Co rts et al. (2013) made each BF from PVC (Polyvinyl Chloride) pipe of 2 m tall and 0.185 m of internal diameter. The bio-filtration column packed with agave fiber was 1 .80 m tall. The characteristics of the influent and its flow ra te play an important role in the performance of the filter. The inffluent of Ria hi et al. (2009) was introduced into the reactors in a constant flow rate ranging from 1 1.8 to 72 mL min using domestic

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29 wastewater from the secondary effluent of an activa ted sludge treatment plant. Vigueras-Corts et al. (2013) used wastewater flow rates ranging from 5 to 25 mL min with wastewater collected after primary treatment of a treatment plant. 2.10.2 The start-up period The start-up period is a fact that determines the p erformance of the biofilters. It is the period of time to grow the biofilm, provi ded to wastewater treatment in order to stabilize and routinely control the process (EPA 1973). Rebah, et al. 2010, with the clay and plastic media systems, provided a 2 week s tart-up period to the systems. But some other studies provided even one or two months. Vigueras-Corts, et al. 2013 provided 3 months at a flow rate of 3 mL min for the agave BFs to be conditioned. During the phase right after the start up, sometime s the COD and other pollutant removal were not very efficient. This could be expl ained due the lack of acclimatization of the biomass in the reactor. When this happened, the initial phase forms part of the start-up (Rebah, et al. 2010). 2.10.3 Studies that remove nitrogen effectively There are some packing materials that have been sho wn efficient in the removal of nitrogen. Providing aerobic-anaerobic sc enarios to obtain nitrificationdenitrification processes have recently been applie d to the nitrogen removal. During the nitrogen cycle, three stages are developed; aer obic nitrification, anaerobic denitrification, and nitrogen fixation (Bernat, et al. 2011). Thus, systems with aerobic and anaerobic stages can achieve nitrification-deni trification processes. Removal of nitrogen through nitrification-denitrification has been proven in previous research. Many studies used different methods and/or media to develop nitrificationdenitrification processes by the establishment of a erobic-anaerobic stages.

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30 Bernat et al. (2011) developed a sequencing batch r eactor (SBR) including aerobic and anaerobic phases in the modified cycle. Bernat objective was to evaluate nitrification and denitrification processes at low DO concentration. Papers such as Nozhevnikova, et al. 2012 relates the efficiency of nitrogen removal to the conjugated nitrification, denitrification, and anammox process es. The functioning of nitrification, denitrification, and anammox processes was demonstr ated during laboratory cultivation methods and during previous studies of the processes in batch and continuous reactors. Yuan, et al. 2012 developed a step-feed anaerobic-(oxic/anoxic) Membrane Bioreactor. The complete system consisted of an anaerobic reactor and then multiple phases of aerobic and anoxic zones in sequence, concluding with a continuous aerated membrane bioreactor. However, on ly few studies of nitrogen removal using aerobic reactors in series have been carried out. 2.10.4 Anaerobic filters Anaerobic pretreatments are used to break down biod egradable substances and to decrease organic material. Besides pollution red uction, anaerobic treatments produce energy through methanogenesis. Low sludge p roduction has been achieved in established anaerobic wastewater treatments. In add ition, anaerobic treatments do not require a complicated operation nor high operation costs (Rebah, et al. 2010) because no oxygen is needed. The combination of filter medi a with anaerobic conditions results in total biomass retention, excellent efflu ent quality, low sludge production, small foot print, and energy production (Lin, et al 2013). COD depletion has also been observed in anaerobic conditions (Zeng, et al. 2010). 2.10.5 Aerobic filters In order to provide aerobic conditions, air is inje cted to the bio-filters. The air is usually injected at the bottom of the BF. The ae ration rate may be different for each

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31 experiment. There are some studies that just mainta in the air concentration at some level introducing the sufficient amounts of air. Vi gueras-Corts et al. (2013) used mechanical aeration by injecting air at the bottom of three biofilters of six. The aeration rate was 0.62 m m h . Nitrification-denitrification processes as well as phosphorous removal in high levels have been achieved using only aerobic bio-fi ltration (Rebah, et al. 2010). Bernat et al. (2011) achieved ammonia removal using volumetric exchange rate of 0.1 d , 0.3 d , and 0.5 d during the aeration phases observing ammonia oxid ation through zero order kinetics. The ammonia nitrogen o xidation achieved at the end of the phases was from 96 to 98.4%. DO level is one of the key factors when developing nitrification-denitrification (Zeng, et al. 2010). 2.10.6 Other factors influencing aerobic and anaero bic biofilters Temperature: The temperature of the biofilters can be used as a co-variable of their operation. Rebah et al. (2010) operated their aerobic BFs under room temperature while in the anaerobic BFs the temperat ure was fixed to 35C. Energy must be provided in order to maintain temperatures above room temperature. Thus, operation costs increase. Vigueras-Cortes et al. (2 013) operated the BFs under their natural temperature without providing any energy th at would increase operation costs. pH: A neutral pH seems to be suitable for nitrificatio n and denitrification processes in BFs. Zeng, et al. (2010) reported a p H range between 7.12 and 7.46 in the nitritation and denitritation of domestic waste water. During their study, ViguerasCortes et al. (2013) observed a neutral pH between 6.67 and 7.28 in every step of the process.

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32 2.10.7 Nitrification and denitrification processes using anaerobic and aerobic systems Many wastewater treatments have applied anaerobic-a erobic process to eliminate nitrogen and other pollutants. Some examp les are anaerobic filter-activated sludge system, up-flow anaerobic sludge blanked (UA SB)-attached aerobic filter, anaerobic baffled reactor-activated sludge system, and aerobic-anaerobic filters (Rebah, et al. 2010). Nitrogen removal has been att ributed to the complete nitrification process and denitrification phenomeno n that occurred (Garzn-Zuiga and Buelna 2011). During nitrification, ammonia is oxidized forming nitrates by Nitrobacteria action and nitrites by Nitrosomas action Thus, existing predominant bacteria determine the majority of the nitrogen for m (nitrates or nitrites). When concentration of nitrites is higher than concentrat ion of nitrates, an incomplete nitrification process was performed (Rebah, et al. 2010). To achieve nitrogen removal of wastewater, a partial nitrification to n itrite and then to nitrate must be developed, followed b denitrification (Bernat, et a l. 2011). Denitrification is enhanced because of the presence of anoxic zones that allowe d the growth of heterotrophic bacteria that reduce nitrates to nitrogen gas (Gali l, Malachi and Sheindorf 2009). Highly concentrated wastewater can effectively be t reated using combined anaerobicaerobic processes (Lin, et al. 2014). A start-up pe riod is necessary to obtain excellent organic removal. Rebah et al. (2010) developed two anaerobic-aerobic biofiltration systems using clay (system 1) and plastic media (system 2) as packing material. Each system had two reactors connected in series. Wastewater wa s first pumped to the anaerobic reactor. The effluent of the anaerobic reactor was then introduced to the aerobic one as the influent in order to remove remaining contam inants; they were operated as plug

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33 flow reactors. They obtained removal efficiencies o f 91-98% in BOD, 90% in COD, and 60-70% in nitrogen removal. Therefore, the perf ormance of the aerobic reactor was determined based on the performance of the anae robic reactor which constitutes the first part of the treatment (Lin, et al. 2014).

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34 CHAPTER III METHODS This section reviews in detail the methods used for this study. Specifically, I outline how the anaerobic-aerobic system functions, as well as the packing material used. More precise discussions follow, surrounding the topics of wastewater influent, the start-up period, and experimental procedure bef ore briefly summarizing the analytical methods and experimental design used. 3.1 Hybrid system The system included two reactors connected in serie s, an anaerobic reactor followed by an aerobic performed in duplicate. Each system was operated as plug flow reactors (Vigueras-Corts, et al. 2013). Figur e 3 shows how the raw wastewater was first pumped to the top of the anaerobic reacto rs (BF1 and BF2) with peristaltic pumps MasterFlex Model 751800. After the treatment in the anaerobic reactor, the effluent was fed into the aerobic reactors (BF3 and BF4) to finish with the treatment. Figure 3 Schematic diagrams of the two combined anaerobic-ae robic BF systems

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35 Figure 4 Anaerobic-aerobic BF system The reactors consisted of polyvinyl chloride (PVC) pipe, 2.0 m high with an internal diameter of 0.185 m. The four biofilters ( BFs) were packed with agave fiber that was previously washed and dried according to G arzn-Zuiga et al. (2003). The biofilters had a 1.80m biofiltration column packed with agave fiber. Additionally, four plates were located along each column as internal d ivisions in order to separate the packing material, to prevent compaction, and to avo id plugging or clogging of the fiber. The acrylic plates were perforated to allow wastewater flow and the system was run at room temperature. The aerobic BFs were supplied with an air loading o f 0.62 m m h counter current flow of wastewater. The air supply was moni tored daily using a Dwyer RMA2 flow meter. Pressure drop was also measured using a pipe connected to a hydraulic gauge. The BFs were installed outdoors in order to mimic a natural environment of a possible future installation in the rural municipal ities of Durango. The temperature of the BFs was used as a co-variable.

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36 3.2 Filter material Agave plant has an extensive use in Mexico. It is u sed in the production of medicine and fibers as well as in fermented and dis tilled drinks. There are around 48 species of agave used in spirits production (Ramire z-Tobias, et al. 2012). Agave is a species that can be located in arid and semi-arid r egions in the north of Mexico. The species Agave durangensis is cultivated in Durango (Figure 5). This plant is used on Mescal production, the typical distilled spirit of the region that is an alcoholic beverage similar to tequila. In Durango, 3.8 millio n agave plants were cultivated in 2007 according to the statistics of SEDECO (2008). After mescal production by fermentation and distillation of the plant, the aga ve fiber is considered useless material and is disposed. Figure 5 Agave Duranguensis Plants from arid and semi-arid regions such as agav e can provide a wide variety of organic materials to the treatment of wa stewater. This represents an advantage to these regions due to the scarcity of w ater and the necessity of water

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37 reuse. Agave plants have many advantages; they are able to adapt to dry climates by storing water and growing slow. Agave fiber is an o rganic material readily available, inexpensive, and is also considered a waste of the alcoholic beverage processes (Vigueras-Corts, et al. 2013). Agave fiber wastes can be used to pack columns in t he biofiltration of wastewater. In addition to filtration, this materia l favors the retention of organic matter and pollutants by adsorption, absorption and ion exchange mechanisms (Garzn-Zuiga, Tomasini-Ortiz, et al. 2008). Thus, agave fiber represents a potential organic material in the development of BFs to the t reatment of municipal wastewater. This experiment employed agave fiber from the mesca l industry in Nombre de Dios, Durango. The fiber was transported to the res earch center (Figure 6), washed and dried outside (Figure 8). It was also passed th rough a 100-mesh sieve removing small particles. Figure 6 Agave fiber reception

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38 Figure 7 Agave fiber before washing Figure 8 Preparation of the agave fiber The fiber was analyzed by measuring cellulose, lig nin and acid detergent fiber concentrations. This characterization was made usin g the methods described by Tejada (1985). The aparent density and porosity wer e also measured from a small volume of fiber. Four columns were packed in the co mpartment between the four perforated acrylic plates contained. The packing wa s based on the moisture content of 65% that was previously wetted with tap water. Thes e measurements and procedures

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39 of the packing material were based on the ones pres ented by Vigueras-Corts, et al. (2013) and the references cited therein. Figure 9 Biofilter single column 3.3 Wastewater influent The raw wastewater was collected once every week fr om the Municipal wastewater treatment plant of Durango City. The was tewater was sampled from the plant after the primary treatment, which provides s creening, sedimentation, and homogenization. The sample of wastewater was taken during the first peak massloading (Figure 10). The peak mass-loading refers t o the period of time when wastewater concentration is the highest. For Durang o City, the first peak massloading is between the 12th and 15th hour each day. At this time, the plant receives all the wastes generated during the morning activities. The second peak mass-loading is at the 18th to 20th hour. However, the collection o f the wastewater was only developed during the first peak.

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40 Figure 10 Wastewater collection The wastewater collected was then transported to th e laboratory and properly disposed in a 400 L polyethylene tank. The wastewat er was fed in the BFs with peristaltic pumps (MasterFlex Model 751800). 3.4 Start-up period The start-up period was provided to the BFs after l eak and hole tests on the filter material. During this time, the BFs were eva luated for three months using a flow rate of 5 mL min . The first step ended when the parameters of BOD reach the allowable values for the standard regulations (30 m g L ) and remain stable. No other seed was used for the start-up phase of the BFs. 3.5 Experimental procedure After the start-up period, the flow rate of the eff luent was increased. Four different flow rates were tested; 10, 15, 20, and 2 5 mL min that correspond to four different HRL (Hydraulic loading rates); 0.54, 0.80 1.07, and 1.34 m m d , respectively. The HRL indicated the four phases of the experiment. As it was mentioned, the aeration rate in the aerobic filters was 0.62 m m h. The aerobic

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41 filters were labeled BF1 and BF2. The non-aerated B Fs were labeled BF3 and BF4 (Figure 11). This experiment was conducted within 360 days continuous. Figure 11 Biofilters Columns 3.6 Analytical methods The following parameters were analyzed according to the Standard Methods (APHA 1998). Biological oxygen demand according to the 5-days BOD test method 5210; Chemical Oxygen Demand according to the Close d Reflux, Colorimetric method 5220; Total Solids according to Total Solids Dried at 103-105C method 2540; Fecal Coliform according to Fecal Coliform Pr ocedure method 9221; Nitrates

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42 by capillary electrophoresis with ultraviolet detec tor to 211 nm using a buffer 20 mmol Tris and 20 mmol NaCl. Samples from the influent and effluent of each syst em and each BF were analyzed every week (Figures 13). Electrical conduc tivity (EC) and pH were also measured once a week. Electrical conductivity was m easured according to Conductivity, Laboratory method 2510 also from APHA 1998. Air flow rate internal and room temperature were monitored from Monday to Friday. Figure 12 Effluents of the start-up period Figure 13 Effluents and influent of the last phase

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43 3.7 Experimental design Two variables were considered in the experimental d esign based on the HLRs (0.54, 0.80, 1.07, and 1.34 m m d ) and the aeration (with air and without air). The variables were tested using a factorial experiment. Internal temperatures of the BFs were co-variables in the experiment. The statistica l method and its sources of variation were evaluated with the Statistica softwa re (StatSoft, 2004).

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44 CHAPTER IV RESULTS AND ANALYSIS This section outlines the results obtained in the d ifferent parameters of the study. First, I include a table with the wastewater characterization with the initial concentrations in the influents. Then, final concen trations and removal efficiencies of organic matter, fecal coliforms, total suspended so lids, and nitrates are concisely presented and illustrated. Additional graphs are al so presented to illustrate some of the statistical analyses. 4.1 Wastewater characterization The composition of the raw wastewater that was used in the study is shown in table 4.1. Table 4.1 Average composition of the raw wastewater Parameters Average concentration S.D. Units Sample number Biochemical Oxygen Demand 252 37 mg L 27 Chemical Oxygen Demand 706 165 mg L 29 Total Suspended Solids 62 36 mg L 19 Fecal Coliform 1.26x10 7 2.15x10 7 MPN 100 mL 35 Nitrate 0.0 mg L 38 Electric Conductivity < 850 S cm 31 pH 7.07 0.69 30 S.D. = standard deviation, MPN= most probable numbe r 4.2 Organic matter (BOD and COD) removal effect During the start-up period (HRL=0.27 m m d ), removal of BOD occurred in the aerobic reactors achieving concentrations be low the allowable 30 mg L

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45 (Figure 15). The stat-up period was eighty five day s and ended once BOD concentration in the effluents achieved levels in c ompliance with the regulations and was constant. However, the anaerobic reactors did n ot achieve the permissible levels and they have an increase in BOD concentration up to 150 mg L during the start-up period (Figure 14). These results match with the st atistical analysis which indicates that aerobic BFs provide the highest BOD removal. During the first stage (HLR=0.54 mm d ), effluents from anaerobic reactors were slightly above the regulation but once the aer obic treatment was provided, concentrations below 30 mg L were always achieved. The following two stages of the BFs (HLR=0.80 and 1.07 mm d ) were also able to provide permissible concentrations after the aerobic treatment. The max imum removal efficiency of BOD in the system was 96% with an effluent of only 17 m g L . Figure 14 Effluent BOD concentrations in the anaerobic BFs operated at fo ur different hydraulic loading rates 0 50 100 150 200 250 300 350 400 050100150200250300350BOD5(mg/L)Time (d) BF without air Influent NOM-003-SEMARNAT-1997 0.27 m3/m2d 0.54 m3/m2d 0.80 m3/m2d 1.07 m3/m2d 1.34 m3/m2d

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46 Figure 15 Effluent BOD concentrations in the aerobic BFs operated at four different hydraulic loading rates. The COD had an increase during the start-up period in the anaerobic BFs, reaching a COD concentration of almost 2000 mg L (Figure 17). This might be caused by the dissolution or washing of the caramel ized carbohydrates of the fiber that were attached during the cooking process of th e mescal production. On the other hand, the aerobic BFs started removing COD from the start-up period (Figure 18). The COD concentrations in the effluents decrease fr om 750 to 400 mg L during the start-up period of the aerobic BFs which indicates that aeration promotes fast removal of components. In the first stage (HLR=0.54 mm d ), the COD decreased significantly maintaining COD concentrations below the influent concentrations. During the first stage COD concentrations decreased from 1080 to 250 mg L and from 380 to 130 mg L in the anaerobic and aerobic BFs, respectively. The maximum removal efficiency of COD was 87% at HL R of 1.34 mm d in the aerobic BF. Thus, in the aerated BFs, the CO D had significant reductions. The 0 50 100 150 200 250 300 350 400 050100150200250300350BOD5(mg/L)Time (d) BF air Influent 0.27 m3/m2d 0.54 m3/m2d 0.80 m3/m2d 1.07 m3/m2d 1.34 m3/m2d NOM-003-SEMARNAT-1997

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47 lowest concentration of COD was 50 mg L and was observed at the end of the experiment (HLR=1.34 m m d ). Figure 16 COD average concentrations in the anaerobic biofilt ers Figure 17 COD average concentrations in the aerobic biofilter s 0 500 1000 1500 2000 2500 050100150200250300350COD (mg/L)Time (d) BF without air Influent0.27 m3/m2d 0.54 m3/m2d 0.80 m3/m2d 1.07 m3/m2d 1.34 m3/m2d 0 500 1000 1500 2000 2500 050100150200250300350COD (mg/L)Time (d) BF air Influent 0.27 m3/m2d 0.54 m3/m2d 0.80 m3/m2d 1.07 m3/m2d 1.34 m3/m2d

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48 4.2.1 Statistical analysis of the organic matter re moval efficiency 4.2.1.1 Biochemical Oxygen Demand According to the covariance test the temperature, a ir and air*HLR interaction were statistically significant (p=0.001) for BOD removal. Mean analysis based on the Fishers method showed that the hybrid system had m ore BOD removal efficiency at HLR of 0.54 m m d and the BFs with air effect had more efficiency (Figure 16). Figure 18 Air*HLR interaction on biochemical oxygen demand re moval in the system at four different hydraulic loading rates. Table 4.2 BOD average concentrations in the effluents HSL (m3m-2d-1) BF w/out air (mg L ) S.D. BF air (mg L ) S.D. 0.54 44 27 17 5 0.8 47 12 32 18 1.07 21 16 39 18 1.34 57 17 57 18 S.D. = standard deviation air 1 air 2 0.540.801.071.34HLS (m3m-2d-1) -20 0 20 40 60 80 100 120BOD5 (mg/L)

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49 4.2.1.2 Chemical Oxygen Demand According to the covariance test the intercept, tem perature and air were statistically significant (p=0.001) for COD removal Mean analysis based on the Fishers method showed that the hybrid system had m ore COD removal efficiency at HLR of 1.34 m m d (Figure 20) and the BFs with air effect had the h ighest removal efficiency (Figure 19). Figure 19 Air effect in the Statistical analyses of COD remo val in the hybrid system Table 4.3 COD average concentrations in the effluents HSL (m3m-2d-1) BF w/out air (mg L ) S.D. BF air (mg L ) S.D. 0.54 530 262 299 124 0.80 337 92 129 43 1.07 349 70 130 40 1.34 286 88 128 86 S.D. = standard deviation AirWithout air 100 150 200 250 300 350 400 450COD (mgL-1)

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50 Figure 20 HSL effect in COD removal in the hybrid system 4.3 Fecal coliforms removal effect The hybrid system showed removal of FC even during the start-up period. During the first and last stages (HLR=0.54 and 1.34 m m d ) FC removal efficiencies reached 98 (1 log units) and 99.98% (3 log units) in the unaerated and aerated BFs, respectively (Table 4.4). The best sta ge for FC removal was in the aerobic BFs at HLR of 0.80 m m d (Figure 21), the maxima average FC removal efficiency from the effluent reached 99.8 (2 log un its) and 99.99% (4 log units) in the unaerated and aerated BFs (Table 4.4), respectively When the HLR was 1.07 m m d the FC removal efficiency was 99.98% (3 log units ) in both aerobic and anaerobic BFs. FC removal in the start-up period wa s lower than in the first stages. 0.540.801.071.34 HSL (m3m-2d-1) 150 200 250 300 350 400 450COD (mg/L)

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51 Figure 21 Fecal Coliform concentrations in anaerobic biofilte rs Figure 22 Fecal Coliform concentrations in aerobic biofilters 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 050100150200250300350Fecal coliforms (MPN/100 mL)Time (d) BF Withou air Influent 0.54 m3/m2d 0.80 m3/m2d 1.07 m3/m2d 1.34 m3/m2dNOM-003-SEMARNAT-1997 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 050100150200250300350Fecal coliforms (MPN/100 mL)Time (d) BF air Influent 0.54 m3/m2d 0.80 m3/m2d 1.07 m3/m2d 1.34 m3/m2d NOM-003-SEMARNAT-1997

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52 Table 4.4 Fecal coliform removal efficiencies in logarithmic units at four different hydraulic loading rates HLR (m 3 m 2 d 1 ) BF w/out air (log units) BF air (log units) BF w/out air Removal % BF air Removal % 0.54 1 3 98.83 99.98 0.8 2 4 99.83 99.99 1.07 3 3 99.98 99.98 1.34 1 3 98.83 99.98 4.3.1 Statistical analyses of fecal coliform remova l According to the covariance test, the intercept, ai r, hydraulic loading rate, and air*HLR interaction were statistically significant (p=0.001) for fecal coliform removal. Mean analysis based on the Fishers method showed that the hybrid system had more Fecal Coliform removal efficiency at HLR o f 1.34 m m d (Figure 24) and that the air effect provided the highest remova l efficiencies (Figure 23). Figure 23 Air effect in the Statistical analyses of FC remov al in the hybrid system AirWithout air -1.5E5 -1E5 -50000 0 50000 1E5 1.5E5 2E5 2.5E5 3E5 3.5E5 4E5 4.5E5 5E5Fecal Coliform (MPN/100 mL)

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53 Figure 24 HLR effect of fecal coliform removal in the hybrid system 4.4 Total suspended solids removal effect Total suspended solids concentrations of the efflue nts are shown in figure 23. The highest TSS removal efficiencies were observed when HLR was 1.07 mm d but the lowest TSS average concentration was observ ed at HLR of 0.80 m m d (Table 4.5). They were 80 and 77% in the unaerated and aerated BFs, respectively. However, in the aerobic BFs the TSS removal efficie ncy was 78% when HRL was 0.54 m m d (Table 4.5). Table 4.5 Total suspended solid effluent concentrations and r emoval efficiencies HLR ( m3m-2d-1 ) BF w/out air Effluent concentration (mg L ) S.D. BF w/out air Removal (%) BF air Effluent concentration (mg L ) S.D. BF air Removal (%) 0.54 35 28 47 14 8 78 0.8 22 16 39 13 9 63 1.07 15 8 80 17 7 77 1.34 17 4 75 18 7 73 S.D. = standard deviation 0.540.801.071.34 HSL (m3m-2d-1) 0 50000 1E5 1.5E5 2E5 2.5E5 3E5 3.5E5 4E5Fecal Coliform (MPN/100 mL)

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54 Figure 25 Total suspended solids concentrations in the two bi ofilter series 4.5 Nitrates removal effect Two hybrid systems were tested for NO3 removal. System 1 (wastewater flow from BF1 to BF3) had amore unstable behavior conc erned to nitrates removal. System 2 (wastewater flow from BF2 to BF4) provide the best performance in nitrates removal at HLR of 0.54 and 0.8 m m d . Results for nitrates removal (NO3 -) in system 2 are presented in figure 26. The lowest level of NO3 concentration observed in the systems was an average of 7 mg L at HLR of 0.54 mm d (Table 4.6). The anaerobic BF of system 2 had NO3 effluent concentrations below 10 mg L at HRLs of 0.54 and 0.80 m m d (Figure 26). 0 20 40 60 80 100 120 140 160 180 050100150200250300350Total suspended Solids (mg/L)Time (d) BF air BF without air Influent NOM-003-SEMARNAT-1997 0.54 m3/m2d 0.80 m3/m2d 1.07 m3/m2d 1.34 m3/m2d

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55 Figure 26 Nitrate (NO3 -) concentrations in system 2 from BF2 to BF4 Mean analysis based on the Fishers method showed t hat anaerobic BFs had less NO production at HLR of 0.54 mm d (Table 4.7). The intercept, air, temperature and HLR were statistically significant for NO removal. Table 4.6 NO average concentrations in the effluents HSL (m m d ) BF w/out air (mg L ) S.D. BF air (mg L ) S.D. 0.54 7 3 135 38 0.80 10 5 211 76 1.07 11 2 179 46 1.34 17 8 222 9 S.D. = standard deviation 4.5.1 Statistical analysis of the NO removal According to the covariance test the intercept, air hydraulic loading rate and HLR interaction were statistically significant (p=0 .001) for NO removal. Mean

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56 analysis based on the Fishers method showed that a naerobic BFs (Figure 27) had more NO removal efficiency at HLR of 0.54 mm d (Figure 28). Figure 27 Air effect in the statistical analysis of NO removal in the hybrid system Figure 28 HLR effect of NO removal in the hybrid system Air Without air 0 20 40 60 80 100 120 140 160 180 200 220 NO3 (mg/L) 0.540.801.071.34HSL (m3m-2d-1) 20 40 60 80 100 120 140 160 180NO3(mg/L)

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57 CHAPTER V DISCUSSION This section presents a detailed discussion of the results obtained for this study. Specifically, I review the results of each p arameter and compare them with other studies. Additionally, possible explanations of some behaviors are explained based on literature. 5.1 Organic matter (BOD and COD) removal effect In the BFs without air, the majority of the BOD con centrations were not allowable by the Mexican regulations. However, the aerobic treatment in the aerated BFs was highly successful in the removal of BOD. Th e HLRs from the start-up period to the second stage (0.80 m m d ) provided results below 30 mg/L of BOD. Low BOD removal in the anaerobic BFs at the beginni ng of the experiment can be explained because of the low development of anaerobic biofilm present at the first stage as well as the effect caused by the was hing deficient of the packing material. However, the allowable concentration of B OD was not achieved during the anaerobic stage. The agave fiber BFs obtained lower concentrations o f BOD than Rebah, et al. (2010) that obtained concentrations between 42 and 74 mg L treating synthetic wastewater within two hybrid systems using a second ary sludge from a municipal wastewater treatment plant and temperature of 35 C The agave fiber BFs achieved similar removal effici encies to Garzn-Zuiga and Buelna (2011) that obtained a removal efficency of 97% of BOD using pruning waste from two tree species, mechanical aeration, a nd an average influent of 264 mg L . But, the HLR of the experiment was almost 10 tim es smaller (0.078 m m d ) than the HLRs used in this study (0.54 to 1.34 m m d ).

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58 Comparing BFs with the same structure and organic m edia, Vigueras-Corts, et al. (2013) obtained a BOD of <30 mg L in the columns without air when using HLRs of 0.54 and 0.80 m m d and in the columns with air when using HLRs from 0.27 to 0.80 m m d . The variation in the results can be awarded to t he prewashing and predrying that was provided to the fiber in thi s study. One of the studies that obtained higher removal eff iciencies than agave fiber BFs was Gilbert, et al. (2008) that obtained BOD remova l 95-99% using a three layer biofiltration of 1) pozzolana, 2) mix of wood chips and peat, and 3) mix of peat, wood chips, and calcite. However, the HLR used was very small, 0.017 m m d . Garzn-Ziga (2011) obtained 99% removal of BOD tr eating wastewater from pig farm using chip wood and peat as the filter med ia and HLRs of 0.07, 0.045, and 0.035 m m d . Buelna, Garzon-Zuiga and Moeller-Chavez (2011) obtained 97% of BOD removal using an HLR of 0.78 m m d . This BF showed obstruction problems when working with high HRL. The lack of ob struction problems with high HRL is an advantage for the agave fiber BFs. 5.2 Fecal coliform removal effect The FC removal efficiencies of the aerated BFs met the Mexican and US standards (1,000 MPN/100 mL ) in few cases at HLRs of 0.54-1.07 m m d in the aerobic BFs. The aerated BFs at HLRs of 0.54 and 1. 07 m m d achieved the maxima average FC removal efficiencies of 99.95% (3 log units). The unaerated BFs did meet neither the Mexican nor US regulations in FC. However, the effluents of the anaerobic BFs were always introduced to the aerobic ones, so few of the final effluents were able to meet the regulations. FC rem oval in the start-up period was lower than in the first stages. According to Garzn -Zuiga and Buelna 2011 the FC

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59 removal efficiency starts occurring increasingly on ce the filtering medium is saturated. Other mechanisms might affect such as pr edation by testates amoebae. Although good FC removal efficiencies were achieved the effluents must be disinfected before being used on irrigation or othe r water reuse activity. However, to avoid this process is recommended to provide a tert iary treatment such as polish lagoon, uv radiations or ozone. Nevertheless, these FC removal efficiencies are greater than the efficiencies reported by Tremblay, Lessard and Lavoie (1996) that used a trickling BF and obtain only 56% reduction o f FC. They are also greater than the efficiencies reported by Han and Dague (1997) t hat obtained 66% of coliform reduction in a mesophilic reactor. Additionally, th ere are some authors that did not achieve FC removal such as Shah, et al. (2002) that worked with a wheat straw BF and treated dairy wastewater. Shah, et al. experime n resulted in an increase of FC from 504x10 r cfu/100mL in the influent to 665x10 r cfu/100mL in the effluent. The removal efficiencies of FC in the agave fiber B Fs were similar to GarznZuiga and Buelna 2011 (4 log units) with an HLR of 0.078 m m d , approximately a tenth of the HLR of the present study. Hill, et a l. (2002) also obtained FC removal efficiencies from 97 to 99% with water temperatures between 23 and 32 C and providing UV radiation. The agave BF system did not require other resource more than air injection. This represent and advantage co mpared with other systems. 5.3 Total suspended solids (TSS) removal effect The TSS removal efficiency was better in anaerobic BFs (80%) than in aerobic ones (78%). The high percentage of voids in the fil ter material is related with the TSS removal efficiency of the BFs and allows for effici ent retention of TSS (ViguerasCorts, et al. 2013). The TSS removal efficiencies (77-80%) are low compared with the efficiencies of other parameters, however, the concentration of TSS in the influent

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60 was actually low because of the efficient primary t reatment that is provided to the water in the wastewater treatment plant (WWTP). Des pite the low efficiencies removal, TSS in the effluent of the aerobic BFs mee t the allowable levels in the NOM-003-SEMARNAT-1997 and the EPA regulations (30 m g L of TSS). The TSS removal efficiency was greater than the one obtaine d by Lens, et al. (1994) that obtained only 72% using peat, bark and wood chips a s a filter media. Garzn-Zuiga and Buelna (2011) obtained a TSS remo val efficiency of 95% with no mechanical aeration and with a HLR 10 times smaller than the hybrid system. Vigueras-Corts, et al. (2013) obtained 93.4 and 91 .9% of TSS removal efficiency in the anaerobic and aerobic BFs, respectively using a lmost the same conditions that in this study but no hybrid connection between the BFs However, the TSS concentration in the raw wastewater of Vigueras-Corts (201 67 mg L ) was higher than the concentration of the wastewater used in this study (62 36 mg L ). Thus, the final concentrations of TSS were low and in compliance wi th the regulations. 5.4 Nitrates removal effect The highest removal efficiency of NO was observed in system 2 (from BF2 to BF4) in the majority of the stages. The concentr ations of NO in the effluents of the BF1 and BF2 decreased because of the denitrific ation that occurred in the reactors. Consequently, the concentrations of NO increased in the effluents of BF3 and BF4 due to the nitrification of ammonia-nitrogen. Denit rification occurred when anaerobic conditions were provided to the microorganisms, as pointed out by Busigny, et al. (2013), and not in aerobic conditions by the hetero trophic bacteria as pointed out by Wen and Wei (2011). Thus, low concentrations of NO were obtained because in the influent the majority of nitrogen compounds were NH4 and nitrification was slightly performed. However, the anaerobic stage helped the system to degrade organic

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61 material, and the lowest concentrations of nitrates that were obtained in the BFs were 7 mg L . These NO concentrations are lower than the 110, 130, and 85 mg L measurements obtained by Bernat, et al. (2011) and were obtained by treating a mixture of wastewater in an SBR (Sequencing Batch R eactor) at limited oxygen concentration and with anaerobic sludge digester su pernatant. The NO concentrations are higher than the ones obtained by Galil, Malachi and Sheindorf (2009) in their anaerobic reactor (between 4.8 and 6.2 mg L ) but in their case, they recycled sludge from an anoxic reactor that they im plemented. The obtention of low nitrate concentrations also reflects that in the an aerobic reactors there may be some aerobic spots where nitrification has taken due to the lack of complete hermeticity of the BFs. Nitrification of ammonia-nitrogen was performed in the aerobic BFs. This assumption is evident because of the high productio n of nitrates in the aerobic BFs. The concentrations of nitrates in the aerobic BFs w ere between 135 and 222 mg L in average. Thus, the ammonia remaining from the anaer obic step was transformed in the aerobic step. However, final concentrations of NH4 are needed in order to prove the removal of this component. Li, et al. (2013) also had an increment of NO concentrations at the end of the treatment where wastewater was treated with an SBR that included with 4 tanks of different anaerobic/anoxic/aerobic function. This i ncreased NO concentrations from 2% in the influent to 47% in the effluent of the to tal nitrogen, most likely because the nitrification-denitrification processes were develo ped in order to decrease ammonianitrogen concentrations.

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62 CHAPTER VI CONCLUSIONS AND RECOMMENDATIONS The BF hybrid system packed with agave fiber is sui table in the treatment of municipal wastewater. It can remove pollutants that cause environmental problems and effects on human health. The effluents of the B Fs met the Mexican and U.S. standards for their use in irrigation or their disc harge on water bodies. The wastewater treated within this system can be safely discharged in the environment and used for irrigation without concern that the water is previo usly disinfected or applying tertiary biological treatment. The efficiency of the agave f iber was again demonstrated as well as its availability and economy. The hybrid system showed the highest BOD removal ef ficiency at HLR of 0.54 m m d with an average concentration of 17 mg L in compliance with the NOM-003-SEMARNAT and the USA-EPA 2004 standards. Th e highest COD removal efficiency was at HLR of 1.34 m m d with an average concentration of 128 mg L . In FC the system showed the highest removal efficie ncy at HLR of 0.80 m m d removing 99.99% of the concentration in the influ ent. 13 mg L was lowest average concentration of TSS in the system and was observed at 0.80 m m d . The anaerobic filters produce effluents with low NO concentrations (7 to 17 mg L ) compared with other systems. The best results were observed in the BFs with air with exception of TSS and NO Higher removal efficiency of TSS and NO was observed in the anaerobic BFs. BFs packed with agav e fiber are excellent removers of total suspended solids in compliance with the NOM-0 03-SEMARNAT-1997. Denitrification and nitrification processes were pe rformed. The concentration of NO during denitrification was low but there was a hig h production of NO in the

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63 aerobic stage. High concentrations of NO in the effluents do not solve the problem of pollution with nitrogen components. Thus, it is suggested to change the order of the system in order to lower the NO production at the end of the treatment, placing fi rst, the aerobic BF and then the anaerobic, in order to obtain nitrification-denitrification processes in the correct order and be able to conve rt all nitrogen components into gas nitrogen.

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64 REFERENCES Aguas del Municipio de Durango. "AMD." 2013. http:/ /www.amd.gob.mx/amd/page (accessed October 23, 2013). Ahmed, Zubair, et al. "Nitrication and denitricat ion using biolters packed with sulfur and limestone at a pilot-scale municipal was tewater treatment plant." Environmental Technology 33, no. 11 (2012): 1271–1278. APHA. Standard Methods for the Examination of Water and W astewater. 19th edn. Edited by American Public Health Association/Americ an Water Works Association/Water Environment Federation. Washingto n, DC, USA, 1998. Araujo, D.J., S.M.S. Rocha, M.C. Cammarota, A.M.F. Xavier, and V.L. Cardoso. "Anaerobic treatment of wastewater from the househo ld and personal products industry in a hybrid bioreactor." Brazilian Journal of Chemical Engineering 25, no. 3 (2008): 443-451. Baker, Richard W. Membrane Technology and Applications. 2nd. Menlo Park, CA: John Wiley & Sons, Ltd, 2000. Berman-Franka, Ilana, Pernilla Lundgren, and Paul F alkowski. "Nitrogen xation and photosynthetic oxygen evolution in cyanobacteria." Research in Microbiology 154 (2003): 157–164. Bernat, Katarzyna, Dorota Kulikowska, Magdalena Zie linska, Agnieszka CydzikKwiatkowska, and Irena Wojnowska-Baryna. "Nitrogen removal from wastewater with a low COD/N ratio at a low oxygen concentratio n." Bioresource Technology 102 (2011): 4913–4916. Bilotta, G.S., and R.E. Brazier. "Understanding the influence of suspended solids on water quality and aquatic biota." Water Research 42 (2008): 2849-2861. Biszel, N., and O. Uslu. "Phosphate, nitrogen, and iron enrichment in the polluted Izmir Bay, Aegean Sea." Marine Environment Research 49, no. 2 (2000): 101-122. Blatchley, Ernest R., et al. "Effects of Wastewater Disinfection on Waterborne Bacteria and Viruses." Water Environment Research 79, no. 1 (2007): 81-92. Bresler, Samantha E. "Policy recommendations for re ducing reactive nitrogen from." ENVIRONMENTAL SCIENCE & POLICY 19-20 (2012): 69-77. Buelna, G., and G. Blanger. "Peat-based biofiltrat ion for small municipalities wastewater treatment." Sci. Tech. Eau 23 (1990): 259–264. Busigny, V., O. Lebeau, M. Ader, B. Krapez, and A. Bekker. "Nitrogen cycle in the Late Archean ferruginous ocean." Chemical Geology 362 (2013): 115-130.

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