INHIBITORY IMPACT OF NITRITE ON THE ANAEROBIC AMMONIUM

INHIBITORY IMPACT OF NITRITE ON THE ANAEROBIC AMMONIUM
INHIBITORY IMPACT OF NITRITE ON THE ANAEROBIC AMMONIUM
OXIDIZING (ANAMMOX) BACTERIA: INHIBITION MECHANISMS AND
STRATEGIES TO IMPROVE THE RELIABILITY OF THE ANAMMOX PROCESS
AS A N-REMOVAL TECHNOLOGY
by
José M Carvajal-Arroyo
________________________________
A dissertation submitted to the Faculty of the
DEPARTMENT OF CHEMICAL AND ENVIRONMENTAL ENGINEERING
In partial fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
WITH A MAJOR IN ENVIRONMENTAL ENGINEERING
In the Graduate College
THE UNIVERSITY OF ARIZONA
2013
THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
As members of the Dissertation committee, we certify that we have read the dissertation
prepared by José M. Carvajal-Arroyo entitled Inhibitory impact of nitrite on the
anaerobic ammonium oxidizing (anammox) bacteria: inhibition mechanisms and
strategies to improve the reliability of the anammox process as a N-removal technology,
and recommend that it be accepted as fulfilling the dissertation requirement for the
Degree of Doctor of Philosophy
_____________________________________________________________11/20/2013
James A. Field
_____________________________________________________________11/20/2013
Maria Reyes Sierra-Álvarez
_____________________________________________________________11/20/2013
Robert G. Arnold
_____________________________________________________________11/20/2013
Raina M. Maier
Final approval and acceptance of this dissertation is contingent upon the candidate’s
submission of the copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and
recommend that it be accepted as fulfilling the dissertation requirement
_____________________________________________________________11/20/2013
Dissertation Director: James A. Field
_____________________________________________________________11/20/2013
Dissertation Director: María Reyes Sierra-Álvarez
2
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of the requirements for an
advanced degree at the University of Arizona and deposited in the University Library to
be made available to borrowers under rules of the Library.
Brief quotations of this dissertation are allowable without special permission, provided
that accurate acknowledgment of source is made. Requests for permission for extended
quotation from or reproduction of this manuscript in whole or in part may be granted by
the head of the major department or the Dean of the Graduate College when in his or her
judgment the proposed use of the material is in the interests of scholarship. In all other
instances, however, permission must be obtained from the author.
SIGNED: José M. Carvajal-Arroyo
3
ACKNOWLEDGEMENTS
I would like to express my gratitude to everyone who has contributed to this
work.
It is my honor to sincerely thank my advisors, Prof. Jim A. Field and Prof. Reyes
Sierra, for giving me the opportunity to be part of their research group, for their
everlasting mentorship and their patience. This dissertation would not have been possible
without their expert guidance, effort and enthusiasm. I am truly fortunate to learn from
them and for the time shared.
I wish to acknowledge Dr. Wenjie Sun for his supervision and help during my
first steps of this research.
I would like to express my appreciation to my colleagues in the anammox group,
Dr. Daniel Puyol and Guangbin Li, for their contributions and for providing countless
chances for discussion.
I am highly indebted with the laboratory assistants within the anammox group,
Robert Reitz, Andy Swartout, Ruben Vazquez and Austin Dougless, for their great help
and contribution to daily laboratory tasks.
I am very thankful to my friends and colleagues from the Environmental
Engineering Group, for making me feel like home from my very first day in Tucson, and
for sharing joys and frustrations of the research
My special gratitude to Lila Otero, for her continuous support and
encouragement, without whom this dissertation would not have been possible.
Finally I would like to acknowledge my family and friends back home, who I
always kept very present in my motivations and whose support I always felt very close. I
thank Ricardo Duyos for encouraging me to move to Tucson and enrolling in this
adventure.
4
DEDICATION
A mi abuelo Arroyo
a mis padres y mi hermana
a Lila
5
TABLE OF CONTENTS
ABSTRACT ...................................................................................................................... 16
CHAPTER 1.- INTRODUCTION .................................................................................... 19
1.1.
Nitrogen pollution ................................................................................................. 19
1.2.
Regulations ........................................................................................................... 21
1.3.
Current biotechnologies for nitrogen removal ...................................................... 23
1.3.1. Conventional nitrification-denitrification processes ................................... 25
1.3.1.1. Nitrification ................................................................................ 25
1.3.1.2. Denitrification ............................................................................ 27
1.3.1.3. N removal technologies based on nitrification-denitrification 28
1.4.
Anaerobic ammonium oxidation........................................................................... 33
1.4.1. Morphology and metabolism: ..................................................................... 35
1.4.2. Application and technologies based on Anammox process ........................ 38
1.4.2.1. Biofilm based systems ................................................................ 41
1.4.2.2. Suspended growth: ..................................................................... 43
1.4.2.3. Hybrid growth systems: ............................................................. 44
1.4.3. Inhibition of the anammox process and consequences ............................... 45
1.5.
Acronym list.......................................................................................................... 47
CHAPTER 2.- OBJECTIVES .......................................................................................... 48
2.1.
Aim ....................................................................................................................... 48
2.1.1. Specific Objectives ..................................................................................... 48
CHAPTER
3.-
INHIBITION
OF
ANAEROBIC
AMMONIUM
OXIDIZING
(ANAMMOX) ENRICHMENT CULTURES BY SUBSTRATES, METABOLITES
AND COMMON WASTEWATER CONSTITUENTS ................................................... 50
6
3.1.
Abstract ................................................................................................................. 50
3.2.
Introduction ........................................................................................................... 51
3.3.
Material and methods ............................................................................................ 53
3.3.1. Microorganisms .......................................................................................... 53
3.3.2. Batch bioassays ........................................................................................... 54
3.3.3. Assessment of anammox activity and inhibition ........................................ 55
3.3.4. Analytical methods ..................................................................................... 56
3.3.5. Molecular methods...................................................................................... 57
3.4.
Results and discussion .......................................................................................... 57
3.4.1. Characterization of enrichment cultures ..................................................... 57
3.4.2. Effects of anammox substrates and metabolites ......................................... 59
3.4.3. Effects of common wastewater constituents ............................................... 62
3.4.4. Effects of nitrification inhibitors ................................................................. 67
3.5.
Implications........................................................................................................... 70
CHAPTER 4.- PRE-EXPOSURE TO NITRITE IN THE ABSENCE OF AMMONIUM
STRONGLY INHIBITS ANAMMOX............................................................................. 72
4.1.
Abstract ................................................................................................................. 72
4.2.
Introduction ........................................................................................................... 73
4.3.
Materials and Methods .......................................................................................... 76
4.3.1. Origin of the biomass .................................................................................. 76
4.3.2. Batch bioassays ........................................................................................... 77
4.3.3. Analytical methods ..................................................................................... 82
4.3.4. Assessment of specific anammox activity and inhibition ........................... 82
4.4.
Results ................................................................................................................... 83
4.4.1. Inhibition by NO2- in the presence or absence of NH4+ .............................. 83
4.4.2. Effect of the duration of the pre-exposure period ....................................... 86
4.4.3. Role of the liquid medium pre-incubated with NO2- ................................. 87
4.4.4. Nitric oxide accumulation ........................................................................... 89
7
4.5.
Discussion ............................................................................................................. 93
4.5.1. Influence of physiological state on NO2- inhibition of anammox bacteria . 93
4.5.2. NO accumulation, evidence of disrupted anammox cells........................... 95
4.5.3. Hypothesis for NO2- inhibition ................................................................... 97
4.6.
Implications........................................................................................................... 99
CHAPTER 5.- THE ROLE OF pH ON THE RESISTANCE OF RESTING- AND
ACTIVE ANAMMOX BACTERIA TO NO2- INHIBITION ........................................ 101
5.1.
Abstract ............................................................................................................... 101
5.2.
Introduction ......................................................................................................... 103
5.3.
Materials and Methods ........................................................................................ 105
5.3.1. Origin of the biomass ................................................................................ 105
5.3.2. Batch bioassays ......................................................................................... 106
5.3.3. Continuous bioreactors ............................................................................. 107
5.3.4. ATP extraction and quantification ............................................................ 109
5.3.5. Analytical methods ................................................................................... 109
5.3.6. Assessment of specific anammox activity and inhibition ......................... 110
5.4.
Results ................................................................................................................. 111
5.4.1. The role of FNA on the inhibition of actively metabolizing anammox
bacteria ................................................................................................................ 111
5.4.2. The role of the pH on the inhibition by NO2- pre-exposure (resting cells)114
5.4.3. ATP response to NO2- stress ..................................................................... 118
5.4.4. Long term effect of pH on the stability of anammox bioreactors ............. 121
5.5.
Discussion ........................................................................................................... 126
5.5.1. Short term effect of NO2- inhibition.......................................................... 126
5.5.2. Mode of action of NO2- and FNA ............................................................. 130
5.5.3. The effect of NO2- and pH during continuous operation .......................... 132
5.6.
Implications......................................................................................................... 133
8
CHAPTER 6.- STARVED ANAMMOX CELLS ARE LESS TOLERANT TO NO2INHIBITION................................................................................................................... 134
6.1.
Abstract ............................................................................................................... 134
6.2.
Introduction ......................................................................................................... 135
6.3.
Materials and Methods ........................................................................................ 139
6.3.1. Batch bioassays ........................................................................................ 139
6.3.2. Continuous bioreactors ............................................................................. 140
6.3.3. Analytical methods ................................................................................... 141
6.3.4. Assessment of specific anammox activity and inhibition ......................... 142
6.4.
Results and Discussion ....................................................................................... 143
6.4.1. Effect of starvation on resistance of anammox resting cells to NO2exposure .............................................................................................................. 143
6.4.2. Metabolically active and resting anammox cells respond differently to NO2inhibition after starvation .................................................................................... 147
6.4.3. Effect of the starvation on the intensity of the response to NO2- stress .... 150
6.4.4. Effect of sustained underloading on anammox bioreactors ...................... 151
6.4.5. Why does starvation affect the tolerance of anammox cells to NO2-? ...... 156
6.5.
Implications......................................................................................................... 158
CHAPTER 7.- CONCLUSIONS .................................................................................... 159
APPENDIX A. Supplementary data for CHAPTER 3 ................................................... 168
APPENDIX B. Supplementary data for CHAPTER 4 ................................................... 173
APPENDIX C. Supplementary data for CHAPTER 5 ................................................... 176
APPENDIX D. Supplementary data for CHAPTER 6 ................................................... 178
REFERENCES ............................................................................................................... 180
9
LIST OF FIGURES
Figure 1.1. Microbial nitrogen cycle. *Most of the nitrogen in biomass is in the form of
ammines, nevertheless, due to contribution of nitrogen from nucleotides, the average
oxidation state of nitrogen in biomass is higher than –III. Adapted from Colasanti, 2011.
DNRA: Dissimilatory nitrate reduction to ammonium. Assimilative processes not
included. ............................................................................................................................ 24
Figure 1.2. Simplified schemes of suspended growth systems for N removal. RAS:
Return Activated Sludge, AD: Anaerobic Digester. ......................................................... 30
Figure 1.3. Simultaneous nitrification and denitrification in microniches of granular
sludge in an aerobic reactor. ............................................................................................. 31
Figure1. 4. Morphology of ANAMMOX microorganisms.............................................. 36
Figure 1.5. Partial nitritation and anammox occurring in microniches of a granule. ...... 42
Figure 3.1. Effect of pH on the anammox activity of a granular enrichment culture (●)
and a suspended enrichment culture (○). .......................................................................... 58
Figure 3.2. Effect of anammox reaction substrates and products on the anammox
activity of a granular enrichment culture (■) and a suspended enrichment culture (□)
exposed to: (a) NH4+, (b) NO2-, (c) NO3-, and (d) N2H4. .................................................. 60
Figure 3.3. Effect of H2S on the anammox activity of a granular enrichment culture (♦)
and a suspended enrichment culture (◊)............................................................................ 64
Figure 3.4. Effect of O2 on the anammox activity of a granular enrichment culture (●)
and a suspended enrichment culture (○). .......................................................................... 66
10
LIST OF FIGURES - continued
Figure 4.1. Experimental protocols for addition of substrates in batch bioassays. .......... 81
Figure 4.2. Impact of NO2- pre-exposure on NO2- inhibition. A, Time course of N2
production with NO2- pre-exposure for 24 h ( ) and with simultaneous NO2- and NH4+
feeding ( ). The NO2- and NH4+ concentrations used were 100 and 38 mg N L-1,
respectively. B, The effect of NO2- concentration on the nSAA after pre-exposure for 24
h to NO2- alone ( ) or when simultaneously fed with NH4+ and NO2- ( ). The
maximum SAA in simultaneously fed treatments and pre-exposed treatments, was
0.33±0.02 and 0.40±0.02 g N g VSS-1 d-1, respectively .................................................. 85
Figure 4.3. Effect of the time of pre-exposure to 100 mg NO2--N L-1 on nAA of
anammox sludge in absence of NH4+ The SAA of the non-pre-exposed control was
0.98±0.02 g N g VSS-1 d-1. ................................................................................................ 86
Figure 4.4. The nAA of simultaneously fed biomass (A), biomass pre-exposed to NO2(B), biomass washed after biomass pre-exposure (C), and healthy biomass exposed to a
medium decanted from another NO2- pre-exposed assay (D). Bars indicate the buffer
system used in each experiment: HEPES buffer ( ), phosphate buffer ( ). The SAA of
the simultaneous fed controls (A) were 0.80±0.03 and 0.92±0.04 g N g VSS-1 d-1 in
HEPES and phosphate buffer, respectively. ...................................................................... 88
Figure 4.5. - Time course of N2 production (closed symbols) and NO accumulation
(open symbols) at initial NO2- concentrations of 57 (diamonds), and 500 (squares) mg
NO2--N L-1 when a simultaneous feeding protocol of NO2- and NH4+ was utilized. ........ 90
Figure 4.6. NO gas accumulation after 24 h of pre-exposing the anammox biomass to
different concentrations of NO2- in the absence of NH4+.................................................. 91
11
LIST OF FIGURES - continued
Figure 4.7. Time course of N2 (A) and NO (B) produced after pre-exposing the
anammox biomass to 100 mg NO2--N L-1 for 0 min (●), 15 min (♦), 1h (▲) and 12 h (■) .
.......................................................................................................................................... 92
Figure 5.1. Effect of Total NO2- (Panel A) and FNA (Panel B) on the anammox activity
at pH = 6.6 ( ), pH = 7 ( ). pH = 7.3-7.4 (∆) and pH = 7.7-7.8 () in metabolically
active cells fed NH4+ and NO2- simultaneously. ............................................................. 113
Figure 5.2. Time course of N2 production by metabolically active anammox cells
simultaneously exposed to NH4+ (76 mg N L-1) and NO2- (100 mg N L-1) at pH 7.03 (▲)
and 7.52 ( ), and NO2- pre-exposed resting cells (100 mg N L-1) subsequently
supplemented with NH4+ (76 mg N L-1) at pH 7.11 ( ) and 7.52 ( ). ......................... 115
Figure 5.3. Effect of the pH on the response of anammox granular sludge to NO2exposure. A, SAA of metabolically active biomass simultaneously exposed to NO2- (100
mg N L-1) and NH4+ (76 mg N L-1) (closed symbols) and SAA of resting cells preexposed for 24h to NO2- (100 mg N L-1) subsequently supplemented with NH4+ (76 mg
N L-1) in bicarbonate buffer (squares) and HEPES buffer (circles). B, Accumulation of
NO gas in the headspace after 24h of pre-exposure to NO2- only (100 mg N L-1) at
different pHs, with HEPES buffer (circles) and bicarbonate buffer (squares). Transient
accumulation of trace NO was also observed during incubation of metabolically active
cells simultaneously fed NO2- and NH4+ (data not shown)............................................. 117
Figure 5.4. Time course of N2 production (close symbols) and evolution of the ATP
content (open symbols) of resting cells pre-exposed to 50 mg NO2--N L-1(A) and 38 mg
NH4+-N L-1 (B) at pH 6.5 (squares), 7.2 (circles) and 8.3 (triangles). The dotted vertical
line represents the addition of the missing substrate, in stoichiometric concentration. .. 120
12
LIST OF FIGURES - continued
Figure 5.5. Effect of the pH on the performance of UASB reactors subjected to NO2and NH4+ substrate interruption, respectively. Evolution of the daily N2 production (A)
and effluent pH (B) of the reactors R1 ( ), R2( ), R3( ) during different operation
periods (I: start up, II: application of different pH to each reactor, III: recovery of
optimum pH in R1). ........................................................................................................ 123
Figure 5.6. Concentration of NH4+ (A) and NO2- (B) in the influent (close symbols) and
effluent (open symbols) of the reactors R1 (diamonds), R2 (squares) and R3 (circles),
during different operation periods (I: start up, II: application of different pH to each
reactor, III: recovery of optimum pH in R1). .................................................................. 125
Figure 6.1. Time course of N2 production of anammox granules pre-exposed for 24 h to
50 mg NO2- - N L-1 (triangles), 38 mg NH4+ -N L-1 (squares), or simultaneously fed
(circles) after a starvation period of 0 d (close symbols) or 26 d (open symbols). ......... 144
Figure 6.2. SAA biomass pre-exposed to NO2- (50 mg N L-1) ( ), NH4+ (38 mg N L-1)
( ) or simultaneously fed with NO2- and NH4+ ( ), after different periods of starvation.
......................................................................................................................................... 146
Figure 6.3. Effect of starvation on the activity of anammox cells exposed to NO2- in
presence of NH4+ (A) or pre-exposed to NO2- (B). Closed symbols represent fresh
biomass. Open symbols represent biomass starved for 20 d (A) or 14 d (B). ................ 148
Figure 6. 4. Evolution of the ATP content of anammox cells during a treatment of
exposure to 100 mg NO2- -N L-1, after a starvation period of 0 d ( ) or 19 d ( ). ...... 151
13
LIST OF FIGURES - continued
Figure 6.5. Influence of NLR on the performance of anammox reactors subjected to
events of substrate interruption. A: Profiles of NH4+ and NO2- concentrations in the
influent and effluent of R2. NO2-inf (solid line), NH4+inff (dashed line), NO2-eff ( ) and
NH4+eff ( ). B: Evolution of the SAA of the biomass of the R1 ( ), R2 ( ) and R3 (▲).
......................................................................................................................................... 155
14
LIST OF TABLES
Table 1.1. Secondary treatment standards for municipal wastewater in the US ............. 22
Table 1.2. Requirements for discharges from urban wastewater treatment plants to
“sensitive areas” subject to eutrophication in the EU. ...................................................... 23
Table 1.3. Reported data about NO2- inhibition of the ANAMMOX process. ................ 46
Table 3.1. Effects of various common wastewater contaminants and nitrification
inhibitors on the activity of a suspended- and a granular anammox enrichment culture 69
Table 4.1. Summary of conditions applied to each experiment ....................................... 80
Table 5.1. pH of the influent of the anammox bioreactors during the different operation
periods ............................................................................................................................. 108
Table 6.1. Reported data about nitrite toxicity on anammox bacteria ........................... 137
15
ABSTRACT
The anaerobic oxidation of ammonium (anammox) with nitrite as electron
acceptor is a microbial process that generates nitrogen gas as main final product. After
being discovered in the Netherlands in the 1990s, anammox has been applied in state-ofthe-art biotechnologies for the removal of N pollution from ammonium rich wastewaters.
The anammox process offers significant advantages over traditional nitrificationdenitrification based processes. Since anammox does not need elemental oxygen, it
allows for important savings in aeration. Furthermore, due to the autotrophic nature of the
bacteria, anammox does not require external addition of electron donor, often needed in
systems with post-denitrification. Although the anammox bacteria have high specific
activity, they are slow growing, with doubling times that can range from 10 to 25 d.
Therefore, in case of a toxic event causing the death of the biomass, a long recovery
period will be required to reestablish full treatment capacity.
The purpose of this work is to investigate the inhibition of anammox bacteria by
compounds commonly found in wastewaters, including substrates, intermediates and
products of the anammox reaction. Among common wastewater constituents, sulfide was
shown to be especially harmful, causing complete inhibition of anammox activity at
concentrations as low as 11 mg H2S L-1. Dissolved oxygen was moderately toxic with a
16
50% inhibiting concentration of 2.3 and 3.8 mg L-1 to granular and suspended anammox
cultures, respectively. Among the various compounds involved in the anammox reaction,
special attention was paid to nitrite. Numerous literature reports have indicated inhibition
of anammox bacteria by its terminal electron acceptor. However to date, there is no
consensus explanation as to the mechanism of nitrite inhibition nor on how the inhibition
is impacted by variations in the physiological status of anammox cells. The mechanisms
of anammox inhibition by nitrite were thoroughly investigated in batch and continuous
experiments of this dissertation. The results of this work demonstrate that conditions
hindering generation of metabolic energy have a detrimental effect on the tolerance of
anammox cells to toxic levels of nitrite. The absence of ammonium during events of
nitrite exposure was shown to exacerbate its toxic effect. As a result of nitrite inhibition,
nitric oxide, an intermediate of the anammox reaction, accumulated in the head space of
the batch experiments. Moreover, nitrite inhibition was enhanced at the lowest range of
pH tested (6.4-7.2), while same nitrite concentrations caused no inhibition under mildly
alkaline conditions (7.5-7.8). Although other authors have relied on the classic concept
that undissociated nitrous acid is the species responsible for the inhibition, the results in
this work indicate that the pH affects the inhibitory effect of nitrite, irrespective of the
free nitrous acid concentration.
Nitrite stress triggered an active response of the anammox bacteria, which
temporarily increased their ATP content to mitigate the inhibition. Additionally,
17
starvation of anammox microorganisms, caused during storage or by sustained
underloading of bioreactors, was found to limit the capacity of the bacteria to tolerate
exposure to nitrite.
The results of this dissertation indicate that the tolerance of anammox bacteria to
NO2- inhibition relies on limiting its accumulation in sensitive regions of the cell. Active
metabolism in presence of NH4+ allows for active consumption of NO2-, avoiding
accumulation of toxic intracellular NO2- concentrations. Furthermore, secondary active
transport proteins may be used by anammox bacteria to translocate nitrite to nonsensitive compartments. Nitrite active transport relies on a proton motive force.
Therefore, conditions such as low pH (below 7.4) or absence of energy sources, which
may disturb the maintenance of the intracellular proton gradient, will increase the
sensitivity of anammox cells to NO2- inhibition. Strategies for the operation and control
of anammox bioreactors must be designed to avoid exposure of the biomass to nitrite
under the absence of ammonium, low pH or after periods of starvation.
18
CHAPTER 1
INTRODUCTION
1.1
Nitrogen pollution
Nitrogen (N) is a necessary element for all living beings. It is a constituent in
DNA, proteins, chlorophyll, etc. In bacterial cells, N accounts for 12% of the dry weight
(Metcalf et al., 2003). Although N is the most abundant element in the atmosphere, most
of it is not available to living organisms since it is present as the inert N2 gas. Only a few
specialized bacteria are able to fix N transforming it to reactive nitrogen (Nr) (Fig. 1.1).
In pre-industrial times, N cycling resulted in a relatively constant global pool of readily
available N. However, the global N cycle has been significantly altered, especially since
1960s, due to human activity (Galloway et al., 2003). In particular, the invention of the
Haber Bosch process, a widely applied method that allows the chemical synthesis of
ammonia from hydrogen (H2) and N2 gas (Smil, 2001), has had an enormous impact on
19
the anthropogenic input of Nr. The amount of Nr fixed has increased from 15 Tg Nr yr-1
in 1860, to 187 Tg Nr yr-1 in 2005, when the Haber Bosch process generated 121 Tg Nr
(Galloway et al., 2008). Although part of the anthropogenic N pollution is dispersed
(from the application of fertilizers for agriculture, combustion of fossil fuels, etc.), a large
share of the fixed N ends up in concentrated streams. Ammonium (NH4+), generated by
mineralization of organic matter, is an important contaminant in a variety of wastewaters,
e.g. domestic wastewater, animal wastes (Driscoll et al., 2003), or landfill leachate (Berge
et al., 2005). Some of this concentrated Nr is discharged in watersheds, i.e. wastewaters
contribute 36-81% of the Nr load to all the estuaries in the north east of the US (Driscoll
et al., 2003). The concentration of nitrate (NO3-), from fertilizers and oxidation of NH4+
pollution, in major rivers in Northeastern U.S. has increased by 3- to 10-fold since
records are available (Vitousek et al., 1997). N-contamination of groundwater has also
been observed in agricultural regions around the globe. Although the groundwater
contamination problem is probably less significant than in surface waters, the long
residence time of water in aquifers means that N contamination is likely to last for a long
time (Howarth et al., 1996).
Since N is a limiting nutrient in uncontaminated water bodies, pollution of water
bodies generates excessive growth of primary producers, also called eutrophication.
When nutrients become limiting and water is too turbid to allow passage of sunlight, the
algae die and degrade, leading to depletion of dissolved oxygen, bad odor and, ultimately,
20
death of fish and loss of biological diversity (Howarth, 1991). NH4+ is toxic to aquatic
life (Randall, Tsui, 2002), and can be oxidized to nitrite (NO2-) and NO3-, consuming the
dissolved oxygen (Karrman and Jonsson, 2001) and causing acidification (Schindler et
al., 1985). The depletion of dissolved oxygen (DO) caused by N pollution has caused the
generation of oxygen-minimum zones like in the Chesapeake Bay, the Baltic and Black
Seas, and the northern Gulf of Mexico (Camargo, Alonso, 2006).
Moreover, NO2- and NO3- pose a threat for human health at concentrations higher
than 10 mg·L-1 (U.S. Public Health Service standard). NO3- in drinking water is
transformed to NO2- in the stomach, which substitutes oxygen in hemoglobin, disturbing
the
transport
of
oxygen
in
the
blood.
This
phenomenon
is
known
as
“methemoglobinemia” and it can cause death in infants (Lee, 1970).
1.2
Regulations
Given the serious threat that nitrogen pollution poses on environment and water
resources legislative measures have been taken in different countries to minimize
discharge of N polluted effluents to water bodies.
21
Table 1.1. Secondary treatment standards for municipal wastewater in the US.
Parameter
30 day Average
7 day Average
BOD5
-1
30 mg L
45 mg L-1
TSS
30 mg L-1
45 mg L-1
Removal efficiency
85% (BOD5 and TSS)
-
pH
6.0-9.0
In the United States, there is not a federal law limiting nitrogen discharges to
surface water. The Federal water pollution control act (1972), requires secondary
treatment for municipal wastewaters (or treatment with comparable effluent quality),
providing effluents which comply with the secondary treatment standards (Table 1.1).
The Clean Water Act (1977) enables states and municipalities to regulate the discharge of
polluted effluents to “waters of the United States”. The regulation of N polluted
wastewaters is applied through the national permit discharge elimination system
(NPDES). The NPDES targets point sources, including publicly owned treatment plants
(public sewage treatment plants), industrial wastewaters, process and cooling water, as
well as stormwater discharges, including both stormwater sewer systems and industrial
sources. Due to the toxicity of NO2- and NO3- to humans, the EPA has set a maximum
contaminant level of 10 mg N L-1 (NO2- + NO3-) in drinking water (National Primary
Drinking Water Regulations), with a maximum NO2- level of 1 mg N L-1.
22
Table 1.2. Requirements for discharges from urban wastewater treatment plants to
“sensitive areas” subject to eutrophication in the EU.
Parameter
Total N
Concentration
Minimum percentage
reduction of total influent
load
15 mg N L-1
(104-105 p.e.)
80
-1
10 mg N L
>(105 p.e.)
Reference method
measurement
Molecular absorption
spectrophotometry
70-80
*Commission Directive 98/15/EC of 27 February 1998, amending Council Directive 91/271EEC. Daily
L-1
; p.e.: population equivalent
average concentration must not exceed 20 mg N
In countries of the European Union, regulation is provided by the Council
Directive on Urban Waste-water Treatment (91/271/EEC). The law sets requirements for
discharges of urban wastewaters and industrial wastewaters. It discriminates between
discharge to “sensitive areas” which are subject to eutrophication (Table 1.2) and
discharge to “less sensitive areas”. The directive requires secondary treatment (or
equivalent) for all wastewater entering collection systems, although for wastewaters
discharged in “less sensitive coastal areas”, primary treatment may be sufficient.
1.3
Current biotechnologies for nitrogen removal
Existing biotechnologies allow for nitrogen removal from wastewaters by taking
advantage of N biotransformation reactions in the microbial nitrogen cycle (Fig. 1.1).
23
+V
NO3-
NITROGEN OXIDATION STATE
+IV
+III
NO2-
DENITRIFICATION
NITRIFICATION
NO
+II
N2O
+I
DNRA
0
-I
N2
ANAMMOX
NH2OH
-II
-III
N2H4
NITRIFICATION
BIOMASS
NH4+
AMMONIFICATION
Figure 1.1. Microbial nitrogen cycle. *Most of the nitrogen in biomass is in the
form of ammines, nevertheless, due to contribution of nitrogen from nucleotides,
the average oxidation state of nitrogen in biomass is higher than –III. Adapted
from Colasanti, 2011. DNRA: Dissimilatory nitrate reduction to ammonium.
Assimilative processes not included.
Even though nitrogen removal has been traditionally accomplished through the
well-known process of nitrification-denitrification, in the last years novel technologies
have been developed which are more cost-effective and allow for treatment of higher N
loading rates than the traditional process. Both, traditional and novel technologies will be
introduced in this chapter.
24
1.3.1 Conventional nitrification-denitrification processes
The nitrification-denitrification process is carried out in two metabolic steps, first
NH4+ is oxidized to NO3- in a strictly aerobic process, and NO3- is then converted to N2
gas. Nitrifying bacteria and archaea play a key role in the microbial cycling of fixed
nitrogen, in seas, estuaries and soils (Mosier et al., 2008)
1.3.1.1 Nitrification
Nitrification is the chemolitoautotrophic aerobic oxidation to NO3- of nitrogenous
inorganic compounds, such as NH4+, hydroxylamine (NH2OH), and NO2- (Wong, 2003).
Complete nitrification occurs in two stages. First, NH4+ is oxidized to NO2-, and nitrite is
then transformed into nitrate NO3-. Each conversion is carried out by different genus of
bacteria. Ammonia oxidation is carried out by Nitrosomonas, Nitrosococus, Nitrosopira,
Nitrosovibrio and Nitrosobolus (Wong, 2003), also called ammonia oxidizing bacteria
(AOB), with NH2OH as reaction intermediate (Güven, Schmidt, 2009). The equation for
ammonia oxidation by Nitrosomonas, assuming a cell yield of 0.15 g cells/g NH4+-N, is
as follows:
25
NH 4 + 1 .3818 O 2 + 0 .0909 HCO −3 →
→ 0 .0182 C 5 H 7 NO 2 + 0 .9818 NO 2− + 1 .0364 H 2 O + 1 .89 H 2 CO 3
(1.1)
Nitrite oxidation to nitrate is carried out by a diverse group of alpha
Proteobacteria called nitrite oxidizing bacteria (NOB), including Nitrospira, Nitrospina,
Nitrosococcus, and Nitrocystis, however, the most recognized genus is Nitrobacter
(Wong, 2003), whose nitrite metabolism for a cell yield of 0.02 g cells/g NO2--N follows
the reaction:
NO 2− + 0 .0025 NH 4+ + 0 .01 HCO 3− + 0 .01 H + + 0 .4875 O 2 →
→ 0 .0025 C 5 H 7 NO 2 + 0 .0075 H 2 O + NO 3−
(1.2)
By addition of the two processes including cell synthesis, the overall equation is
represented as follows:
NH 4+ + 1 .83O 2 + 1 .98 HCO 3− → 0 .021C 5 H 7 NO 2 +
+ 0 .98 NO 3− + 1 .041 H 2 O + 1 .88 H 2 CO 3
(1.3)
The process is pH dependent, with declining activity below pH 7.0, and consumes
alkalinity, thus, extra CaCO3 is commonly supplemented to avoid pH inhibition (Metcalf
26
et al., 2003). Due to the low growth rate, typical sludge retention time (SRT) in activated
sludge systems is between 10-20 d (Metcalf et al., 2003).
1.3.1.2 Denitrification
Denitrification is the biological reduction of NO3- and NO2- to N2 gas, generally
carried out throughout a heterotrophic process under anoxic-anaerobic conditions (Wong,
2003). Although the complete reduction involves successive reductions with NO2-, nitric
oxide (NO), and nitrous oxide (N2O) as intermediates, it can be performed by a single
kind of bacteria. Different gram-negative Proteobacteria are able to use NO3- and NO2- as
electron acceptors and a wide range of organic compounds as electron donor and carbon
source, to obtain energy, yielding N2 gas as main product. Due to the metabolic diversity
of denitrifying bacteria, they occupy very diverse niches. In the process, alkalinity is
generated as shown in the following equation, assuming a cell yield of 0.45 g cells/g
NO3--N, with methanol as electron donor:
NO 3− + 1 .08 CH 3 OH + 0 . 24 H 2 CO 3 → 0 .056 C 5 H 7 NO 2 +
+ 0 .47 N 2 + 1 .68 H 2 O + HCO 3−
(1.4)
27
Some examples of heterotrophic denitrifiers are Pseudomonas, Alcaligenes,
Paracoccus, and Thiobacillus.
1.3.1.3 N removal technologies based on nitrification-denitrification
Different technologies based on the combination of nitrification and denitrification have
been developed to treat nitrogen from wastewaters. Different configurations of suspended
growth systems are widely used (Fig. 1.2). These processes combine N removal with
elimination of organic matter, and they alternate aerobic tanks, where nitrification occurs,
with anoxic tanks or zones where the NO3- produced in aerated zones is consumed by
denitrifiers using organic matter. In systems with pre-denitrification, the organic carbon
of the wastewater is used as electron donor. On the other hand, in systems with postdenitrification, externally added electron donor is often needed. Bioaugmentation can be
used to enhance nitrification in highly loaded plants, e.g. Bioaugmentation batchenhanced (BABE). This configuration allows for improved denitrification since the size
of the aerobic zone can be reduced (Salem et al., 2002, Salem et al., 2004)
In schemes where nitrogen treatment efficiency is correlated to internal recycle
(e.g. Modified Ludzack Ettinger, Bardenpho), high removal rates are associated to high
reactor volumes and, therefore, to high immobilized costs (Baeza et al., 2004). When
28
post-denitrification is required, additional electron donor may be needed (Zhu et al.,
2008). Furthermore, high oxygen inputs are required for complete nitrification
(BODNH4+= 4.57 kg O2/kg NH4+-N). Conventional plants are not conceived for nitrogen
removal and they are not cost effective for wastewaters with low C/N ratio where high
costs are related to electron donor supply (Tam et al., 1992), and additional cost may be
associated to high sludge production rates.
Combined processes where nitrogen removal is accomplished in a single reactor
have been developed. Nitrification and denitrification can be achieved in sequencing
batch reactors (SBR) alternating aeration with anoxic periods (Keller et al., 1997). The
biomass is allowed to settle at the end of each cycle and just a fraction of the supernatant
is extracted in each cycle. The system selects for high settleability biomass, which allows
for higher concentrations of volatile suspended solids (VSS), higher volumetric removal
rates, and generates an effluent with low total suspended solids (TSS), making
unnecessary a secondary clarifier (Munch et al., 1996). Although it is a very reliable
system, the SBR configuration is not suitable for large plants (Zhu et al., 2008).
29
methanol
Eff
clarifier
clarifier
AERATED
ANOXIC
sludge recycle
sludge recycle
AD
AD
POST-DENITRIFICATION
2 sludges
NO3 Eff
Inf
ANOXIC
PRE-DENITRIFICATION
Modified Ludzack-Ettinger
AERATED
RAS
AD
NO3cla rifier
Inf
AERATED
ANOXIC
Eff
AERATED
ANOXIC
RAS
AD
PRE-DENITRIFICATION
Bardenpho
cl a rifier
Eff
ANOXIC
Inf
AEROBIC
AD
Inf
cl arifier
Oxidation Ditch
Eff
AERATED
Sludge Recycle
BABE
AERATED
Digestate
ANAEROBIC
DIGESTION
Dewatered
Sludge
ENHANCED NITRIFICATION
BABE
Figure 1.2. Simplified schemes of suspended growth systems for N
removal. RAS: Return Activated Sludge, AD: Anaerobic Digester.
30
Simultaneous nitrification and denitrification (SND) can be performed in a single
reactor with granular sludge (Beun et al., 2001, de Kreuk et al., 2005). Application of low
DO concentration allows for formation of gradients in granules enabling for the
generation of aerobic and anoxic microniches suitable for the coexistence of nitrifiers and
denitrifiers in the granules (Pochana, Keller, 1999, Third et al., 2003) (Fig 1.3). The
process has significant advantages since an anoxic tank is not needed and extra carbon
addition can be saved (Zhu et al., 2008).
BOD
CO2
Aerobic
zone
O2
Anoxic
zone
N2
CO2
NH4+
NO3DO
Gradient
Figure 1.3. Simultaneous nitrification and denitrification in microniches of
granular sludge in an aerobic reactor.
31
A more refined process was developed in the Netherlands in which NH4+ is
partially oxidized to NO2- instead of NO3- and the denitrification occurs with NO2- as
electron acceptor (Hellinga et al., 1998). The process was originally called single reactor
system for high ammonia removal over nitrite (SHARON), although later on, SHARON
has been referred to the process of partial nitritation to NO2-, excluding the step of
denitrification. In order to avoid the generation of NO3-, the activity of NOB needs to be
suppressed (Picioreanu et al., 1997). The concentration of free ammonia (NH3), short
sludge retention time, high temperature and limited alkalinity are key parameters that
favor the out-competition of NOB by AOB (Anthonisen et al., 1976, Balmelle et al.,
1992, Hellinga et al., 1998). DO is another important parameter. Although several works
have reported that AOB have higher affinity for O2 than NOB (Picioreanu et al., 1997,
Schramm et al., 2000), this does not seem to be universal, and the selection of the
concentration of dissolved oxygen seems to be case specific (Wett et al., 2013).
Compared to processes involving complete nitrification and denitrification over NO3- ,
SHARON saves 25% oxygen in nitrification, and 40% of the methanol that is often
needed to complete denitrification (Notenboom, 2002).
In the last two decades, the discovery of bacteria able to oxidize NH4+
anaerobically has transformed the perspective of N removal. Utilizing these new bacteria,
novel and more sustainable processes have been developed which will allow the
32
development of energy yielding wastewater treatment plants in the near future (Kartal et
al., 2010a).
1.4
Anaerobic ammonium oxidation
The anaerobic oxidation of NH4+ (anammox) with NO2- as terminal electron acceptor,
yielding N2 and NO3- as main products, is the recently discovered missing link in the Ncycle (Strous et al., 2006). NH4+ was thought to undergo oxidation exclusively under
aerobic conditions until the thermodynamic feasibility of the anaerobic oxidation of NH4+
was predicted in 1977 (Broda, 1977):
NH 4+ + NO2− → N 2 + 2 H 2 O
ΔGº= -359 kJ·(mol NH4+)-1
(1.5)
The occurrence of the process was observed years later in a denitrifying pilot
plant in Rotterdam (The Netherlands) (Mulder et al., 1995), and the biological nature of
the reaction was confirmed by Van de Graaf et al., in 1995
The anammox process is carried out by several chemolithoautotrophic bacteria,
related to Planctomycetes. Five “Candidatus” genera have been studied, Brocadia (Strous
33
et al., 1999a), Kuenenia (Schmid et al., 2001), Scalindua (Kuypers et al., 2003),
Anammoxoglobus (Kartal et al., 2007b) and Jettenia (Quan et al., 2008). Anammox
bacteria are being found worldwide in fresh water ecosystems (Schubert et al., 2006,
Zhang et al., 2007), marine sediments (Rich et al., 2008, Schmid et al., 2007), and
wastewater treatment plants (Mulder et al., 1995), and they are responsible for up to 50%
of the oceanic N losses (Kuypers et al., 2005).
The doubling time of anammox bacteria generally range from 11 to 30 d (Strous et al.,
1998, van de Graaf et al., 1996) although some punctual works have reported doubling
times as low as 1.8 d (Isaka et al., 2006) and 3 d (van der Star et al., 2008). The low
growth rate leads to a low cell yield, as shown by the overall stoichiometry (Strous et al.,
1998):
NH 4+ + 1 .32 NO 2− + 0 .066 HCO 3− + 0 .13 H + →
1 .02 N 2 + 0 .26 NO 3− + 0 .066 CH 2 O 0.5 N 0.15 + 2 .03 H 2 O
(1.6)
Due to the high specific activity of about 0.8 kg N kg dry weight (DW)-1 d-1
(Kartal et al., 2004), the anammox process is emergently used in the treatment of N-rich
wastewaters, especially in cases where C/N ratio is low. In these cases, anammox
presents several advantages over conventional N-removal processes. Due to the
autotrophy of the process, 100% savings are made in organic carbon addition for NO2-
34
and NO3- reduction. Since only half of the NH4+ needs to be partially oxidized to NO2-, up
to 63% of the oxygen supply are saved when compared with the complete nitrificationdenitrification process.
1.4.1
Morphology and metabolism:
The cytoplasm in anammox bacteria is divided in three compartments as shown in
Fig. 1.2. The cell wall is a peptidoglycan lacking, proteinaceous membrane; the first
compartment is called paryphoplasm and it is separated from the riboplasm, where the
nucleoid and ribosomes are, by the intracytoplasmic membrane. Inside the riboplasm,
there is a third and unique, compartment called anammoxosome (van Niftrik et al.,
2008b).
The anammox metabolism occurs in the anammoxosome (van Niftrik et al.,
2004). NO2- is reduced to nitric oxide NO by a NO2--oxidoreductase (NirS), then
hydrazine (N2H4) is formed from NH4+ and NO by a N2H4-synthase enzyme (HZS).
Finally N2H4 is oxidized to produce N2 gas by a N2H4-dehydrogenase (HDH) (Kartal et
al., 2011). A NO2--oxidoreductase oxidizes NO2- to NO3-, generating the electrons
necessary for carbon assimilation (Kuenen, 2008).
35
NH4+
H+
ADP + P
N2
ATP
Riboplasm
NO2H+
Cytoplasmic membrane
Anammoxosome
Paryphoplasm
N2H4
HZS
Intracytoplasmic membrane
HDH
NirS
Anammoxosome membrane
NO
Fe
3e-
1eDNA
Cell wall
glycogen
4e-
H+
ETS
H+
Figure1. 4. Morphology of ANAMMOX microorganisms.
The anammoxosome membrane is unique among microorganisms, since it
contains a very particular type of lipids, called ladderanes (van Niftrik et al., 2004). This
lipids have concatenated cyclobutane rings in the alkyl chain, bound to the glycerol by
ether or ester bonds. The ladderane lipids confer the anammoxosome membrane higher
density and a lower permeability to solutes (Boumann et al., 2009, Damste et al., 2002).
The main function of the anammoxosome is related to generation (Jetten et al.,
2009, Lindsay et al., 2001) and conservation of energy (van Niftrik, 2013). The oxidation
36
of N2H4 in the anammoxosome, generates four high energy electrons shuttled to an
electron transport chain (Jetten et al., 2009, Kartal et al., 2011). The energy released by
these electrons is used to generate an intracellular proton gradient between both sides of
the anammoxosome membrane that energizes the production of ATP in the riboplasm
(van der Star et al., 2010). The low permeability of the anammoxosome membrane would
confer the anammox bacteria a better efficiency in energy conservation, necessary in such
slow growing microorganisms (van Niftrik et al., 2004). Furthermore, it has been
suggested to serve as defensive barrier against highly toxic reaction intermediates (e.g.
N2H4 and NO) (van Niftrik et al., 2004), or against toxic free nitrous acid (HNO2) (Lotti
et al., 2012). The import or export of substrates (NH4+, NO2-) and product (NO3-) into or
from the anammoxosome is regulated by membrane proteins found in its genome, i.e.,
amtB, NirC/focA, NarK (Gori et al., 2011, Medema et al., 2010, van de Vossenberg et
al., 2013).
Additionally, anammox bacteria accumulate glycogen in the riboplasm (van
Niftrik et al., 2008a), which the bacteria would use for cell maintenance during starving
conditions. Moreover, iron particles have been found in the anammoxosome (van Niftrik
et al., 2008a), which could serve as iron reservoir to produce the high amount of heme
proteins necessary for anammox metabolism, or as an alternative electron acceptor when
formate is available as electron donor (Strous et al., 2006).
37
Indeed anammox bacteria have shown metabolically diverse. They can use
electron donors different from NO2- (i.e., NO3-, Fe3+, Mn4+) (Strous et al., 2006).
Moreover anammox bacteria are able to oxidize propionate (Guven et al., 2005, Kartal et
al., 2007b), acetate (Kartal et al., 2008), and formate (Strous et al., 2006) as electron
donors for dissimilative NO3- reduction to NH4+ (Kartal et al., 2007a). The ability of
anammox bacteria to use organic acids expands the perspective of future applications.
Anammox bacteria do not incorporate organic C into cell biomass; therefore the
application of anammox to degradation of C-BOD would be very advantageous over
heterotrophic processes since the sludge production would be significantly reduced. The
application of anammox for C-BOD removal has been already demonstrated at a labscale nitritation-anammox SBR fed with a synthetic wastewater with CH3COOHCOD/NH4+-N of 0.5 (Winkler et al., 2012). Although the results are promising, and the
future applications of anammox may include treatment of sewage (Kartal et al., 2010a)
current applications of the anammox process are limited to N removal.
1.4.2 Application and technologies based on Anammox process
As previously mentioned, the anammox process is cost-efficient compared to
other N-removal processes based on nitrification and denitrification. Anammox
applications are currently restricted to treatment of NH4+ rich wastewaters containing low
38
BOD under mesophilic conditions. It has been applied to treatment of effluents from a
wide variety of sources (Vlaeminck et al., 2012): wastewaters from the food industry fish canning (Dapena-Mora et al., 2006), potato processing effluent (Abma et al., 2010,
Mulder et al., 2012), fermentation and distilleries effluents (Vlaeminck et al., 2012),
glutamate wastewaters (Hu et al., 2013b)-, fertilizer manufacturing industry (Keluskar et
al., 2013), tannery (Abma et al., 2007), semiconductor manufacturing (Tokutomi et al.,
2011), coking effluents (Li et al., 2010), domestic wastewater and digested sludge liquor
(Joss et al., 2009), urine (Udert et al., 2008), digested black water (Vlaeminck et al.,
2009)-, and landfill leachate (Hippen et al., 1997).
The application of the anammox process requires a mixture of NH4+ and NO2- of
approximately 1:1 molar. Since most of N polluted effluents contain NH4+ as the major N
compound, a partial nitritation step is required where half of the NH4+ is aerobically
oxidized to NO2- by AOB. Both processes –partial nitritation and anammox– can be
carried out in separate units or in a single reactor. Both options have distinct advantages
and disadvantages. The two stage partial nitritation-anammox enables the independent
optimization of each step, the risk of anammox bacteria being outcompeted by
heterotrophic denitrifiers is lower than in the single reactor configuration (Hu et al.,
2013a). Furthermore the anammox reaction is inhibited by DO (Egli et al., 2001), which
can be avoided by ensuring anaerobic conditions in the separate anammox reactor. On the
other hand, the immobilized costs and the C-footprint derived from greenhouse gas
39
emissions of the single reactor system are considerably lower (Kampschreur et al., 2008,
Kampschreur et al., 2009). So far, about 40 full scale installations utilize the anammox
process (Hu et al., 2013a), and only four have separate nitritation and anammox reactors
(Desloover et al., 2011, Tokutomi et al., 2011, van der Star et al., 2007).
The industry has adopted the name of deammonification, although other
equivalent terminologies exist for the single stage process, with disregard of the
configuration utilized: Completely autotrophic nitrogen removal over nitrite (CANON)
and oxygen limited autotrophic nitrification-denitrification (OLAND) (Kuai, Verstraete,
1998, Third et al., 2001).
Furthermore, different technologies utilize different commercial terminologies,
namely based on the type of biomass used, and the place where they were developed (Hu
et al., 2013b). Due to the slow growth of anammox bacteria, efficient retention of the
anammox biomass is of utmost importance. The kind of biomass utilized, i.e., biofilm
(attached or granular), suspended, or hybrid, determines the configuration of the reactor,
as well as the control strategy. In all the systems where nitrification needs to be
interrupted in NO2- without further oxidation to NO3-, the repression NOB represents a
challenge (Vlaeminck et al., 2012). Each technology deploys different strategies to avoid
NO2- oxidation, namely based on type of biomass utilized.
40
1.4.2.1 Biofilm based systems
Granular sludge: Granular biomass is utilized both, in two reactor systems, or in single
reactor systems.
Separate SHARON – Anammox: When the partial nitritation and anammox are physically
separated, nitritation by AOBs is performed in an aerated tank (SHARON), without
sludge recycle. Under mesophilic conditions, the doubling time of AOB is shorter than
that of NOB, and short retention time is utilized to avoid NO2- oxidation (Hellinga et al.,
1998). The effluent of the SHARON reactor is decanted in order to minimize
contamination of the anammox reactor with nitrifying biomass. In order to meet the
NH4+:NO2- requirement for anammox, the alkalinity in the SHARON reactor must be just
enough to allow for oxidation of just half of the NH4+ (van Dongen et al., 2001). The
anammox reaction takes place in an upflow anaerobic sludge bed reactor. High upflow
velocity is desired to favor granulation and avoid the washout of the slow growing
anammox bacteria. High volumetric loading rates in the anammox reactor ranging 7.1-9.5
kg N m-3 d-1 have been demonstrated in a full scale plant, with total N removal of up to
90% and NO2- effluent concentrations lower than 10 mg N L-1(van der Star et al., 2007).
Single stage partial nitritation-anammox with granular sludge: Partial nitritation and
anammox can be accomplished in a single aerobic air lift- or sequencing batch- reactor,
41
with granular sludge (Kampschreur et al., 2009, Sliekers et al., 2002, Third et al., 2001).
The creation of aerobic and anoxic microniches allows for growth of AOB on the surface
and anammox bacteria in the core of the granules (Fig. 1.5).
N2
Aerobic
zone
AOB
NH4+
Anoxic
zone
O2
ANAMMOX
NO2-
DO
Gradient
Figure 1.5. Partial nitritation and anammox occurring in microniches of a granule.
The limitation to DO transfer, and consumption of DO by AOB in the outer layer
of the granules allows the maintenance high DO concentrations (5 mg L-1) in the bulk
liquid without causing inhibition to the anammox bacteria (Kampschreur et al., 2009).
Volumetric loading rates of 1.7-2.0 kg N m-3 d-1 with 75% total nitrogen removal, have
been maintained in a full scale plant treating wastewater from a potato processing facility
(Kampschreur et al., 2009).
42
Attached growth biomass: Different technologies utilize plastic carriers as support for
attached growth of anammox biomass (Christensson et al., 2013, Rosenwinkel,
Cornelius, 2005). The dominant configuration for attached growth systems is moving bed
biofilm reactor (MBBR). The diffusional limitations in the biofilm enables for the
generation of anoxic zones were anammox bacteria thrive. Due to the thinness of the
biofilm, low DO concentrations are maintained. NOB repression is achieved by
application of a control loop with online measurement of the ratio between NO3generated to NH4+ consumed. When this ratio is higher than 11% aeration is interrupted
to avoid NOB from utilizing excess DO (Christensson et al., 2013).
1.4.2.2
Suspended growth:
In Switzerland, several wastewater treatment plants utilize anammox to treat digested
sludge centrate. In those plants partial nitritation and anammox occur in single SBR
reactors with continuous aeration. The aeration rate is controlled based on DO (lower
than 1 mg L-1) and NO2- concentration (always lower than 5 mg NO2- -N L-1). The length
of the aeration phase is controlled by online NH4+ measurement (Joss et al., 2009).
Another plant in the Netherlands, treating wastewater from a potato processing
factory, utilizes anammox in a four step N removal process. In this case anammox takes
place in a separate reactor under anoxic conditions, following a partial nitritation reactor.
43
The effluent of the anammox reactor is further polished in two downstream reactors
performing nitrification and denitrification. The performance of the plant is controlled by
the SRT (46 d), provided by a settler installed at the end of the biological treatment train.
The plant operates at a nitrogen loeading rate (NLR) of 0.054-0.066 kg N m-3 d-1 (total
volume) producing a dischargeable effluent (Desloover et al., 2011).
1.4.2.3
Hybrid growth systems:
Deammonification can be carried out in a single stage SBR with suspended biomass
(Wett, 2007). The process is called DEMON®. In order to guarantee appropriate
retention of the anammox biomass hydrocyclones are used to select for high density
granules, enriched in anammox bacteria, which are recycled to the reactor. The reactor is
intermittently aerated. The length of aeration and anoxic periods is controlled based on a
tight pH control. The intermittent aeration causes a metabolic lag-phase to NOB, which
are outcompeted by anammox bacteria (Wett et al., 2010).
Other alternative configurations have been explored in laboratory scale including
rotating biological contactors (Vlaeminck et al., 2012), anammox biomass embedded in
gel carriers (Furukawa et al., 2006) or membrane bioreactors (van der Star et al., 2008).
44
1.4.3 Inhibition of the anammox process and consequences
Due to the slow growth of anammox microorganisms, very long start up periods
are needed. Initial times of 60 d (Dapena-Mora et al., 2004), 58 d (Liao et al., 2007) have
been reported for SBR configurations using activated sludge and methanogenic granular
sludge as inocula. In full scale installations doubling times of 11 d (van der Star et al.,
2007) to 27 d (Joss et al., 2009) have been measured. Strategies including submerged
hollow fiber membrane bioreactors (van der Star et al., 2008) or favoring biofilm
formation (Fernandez et al., 2008) have been reported in the literature aiming to reduce
start-up periods. Nevertheless, exposure to inhibitory conditions could lead to complete
failure of the reactor regardless of the strategy used to retain the biomass. If biomass is
lost due to the presence of toxicants in the wastewater, recovery of the full treatment
capacity would take too long. Little information is available about the susceptibility of
anammox bacteria to compounds commonly found in wastewaters (Dapena-Mora et al.,
2007). Among compounds causing inhibition of anammox bacteria, NO2- , is of special
relevance since it is a necessary substrate of the reaction (Table 1.3). Although numerous
studies have reported inhibition of ANAMMOX bacteria by NO2- , the mechanisms
responsible for NO2- inhibition as well as the operational conditions leading to an
increased risk of failure due to NO2- inhibition are unknown.
45
In this dissertation, the inhibition of the anammox process by compounds
commonly present in wastewaters is investigated. Furthermore, the inhibition of
anammox by NO2- has been thoroughly studied, and insight on the conditions to be
avoided during operation of anammox reactors is provided.
Table 1.3. Reported data about NO2- inhibition of the ANAMMOX process.
NO2- Concn.
Effect
Mode
Conditions
Reference
(mg N L-1 )
100
185
Complete
Inhibition
Complete
inhibition
batch
7-7.8
(Strous et al., 1999b)
batch
7
(Egli et al., 2001)
350
50% Inhibition
batch
7.8
(Dapena-Mora et al.,
2007)
102
Inhibition
SBR
7.5-8.2
(Lopez et al., 2008)
430a,b
37% Inhibition
batch
NR
(Kimura et al., 2010)
60
Inhibition
SBR
7.5
(Fux et al., 2002)
278*
50% Inhibition
batch
7.8
(Fernandez et al., 2012)
500
35% Inhibition
batch
7.6-7.7
(Scaglione et al., 2012)
171-173a
50% Inhibition
batch
7.6-7.7
(Scaglione et al., 2012)
400
50% Inhibition
batch
7.5
(Lotti et al., 2012)
-
Decrease in N
removal
efficiency
Full-scale
gas lift
7-8
(van der Star et al., 2007)
4.8
Decrease in
Activity
Full-scale
Floc-based
SBR
-
(Wett, 2007)
*Calculated from reported data: 11 µg HNO2-N L-1 at pH 7.8; a 24 h pre-exposure-no wash
b
biomass washed after exposure
NR: not reported
46
1.5
Acronym list
AD
AOB
BABE
BOD
CANON
C-BOD
CWA
DNRA
DO
EPA
FWPCA
MBBR
N
NOB
NPDES
Nr
OLAND
SBR
SHARON
SND
SRT
TSS
VSS
Anaerobic Digester
Ammonium Oxidizing Bacteria
Bioaugmentation Batch Enhanced
Biological Oxygen Demmand
Completely Autotrophic Nitrogen Removal Over Nitrite
Carbonaceous Biological Oxygen Demand
Clean Water Act
Dissimilatory Nitrate Reduction to Ammonia
Dissolved Oxygen
Environmental Protection Agency
Federal Water Polution Control Act
Moving Bed Biofilm Reactor
Nitrogen
Nitrite Oxidizing Bacteria
National Pollution Discharge Elimination System
Reactive nitrogen
Oxygen Limited Autotrophic Nitrification-Denitrification
Sequencing Batch Reactor
Single system for High Ammonium Removal Over Nitrite
Simultaneous Nitrification-Denitrification
Solid Retention Time
Total Suspended Solids
Volatile Suspended Solids
47
CHAPTER 2
OBJECTIVES
2.1
Aim
The objective of this research is to investigate the inhibition of the anammox
bacteria by common wastewater constituents, with special attention to nitrite, the terminal
electron acceptor of anammox.
2.1.1 Specific Objectives
1. Evaluate the inhibition of the anammox process by substrates, metabolites and
common wastewater constituents on two different anammox enrichment cultures
48
2. Study the inhibitory effect of nitrite on metabolically active- and resting
anammox bacteria
3. Investigate the role of the pH on the tolerance of anammox bacteria to nitrite
exposure under active and resting conditions.
4. Investigate the effect of starvation on the tolerance of anammox bacteria to nitrite
inhibition.
49
CHAPTER 3
INHIBITION OF ANAEROBIC AMMONIUM OXIDIZING (ANAMMOX)
ENRICHMENT CULTURES BY SUBSTRATES, METABOLITES AND COMMON
WASTEWATER CONSTITUENTS
3.1
Abstract
Anaerobic ammonium oxidation (anammox) is an emerging technology for
nitrogen removal that provides a more environmentally sustainable and cost effective
alternative compared to conventional biological treatment methods. The objective of this
study was to investigate the inhibitory impact of anammox substrates, metabolites and
common wastewater constituents on the microbial activity of two different anammox
enrichment cultures (suspended and granular), both dominated by bacteria from the genus
Brocadia. Inhibition was evaluated in batch assays by comparing the N2 production rates
in the absence or presence of each compound supplied in a range of concentrations. The
50
optimal pH was 7.5 and 7.3 for the suspended and granular enrichment cultures,
respectively. Among the substrates or products, ammonium and nitrate caused low to
moderate inhibition, whereas nitrite caused almost complete inhibition at concentrations
higher than 15 mM. The intermediate, hydrazine, either stimulated or caused low
inhibition of anammox activity up to 3 mM. Of the common constituents in wastewater,
hydrogen sulfide was the most severe inhibitor, with 50% inhibitory concentrations (IC50)
as low as 0.03 mM undissociated H2S. Dissolved O2 showed moderate inhibition (IC50 =
2.3 to 3.8 mg L-1). In contrast, phosphate and salinity (NaCl) posed very low inhibition.
The suspended- and granular anammox enrichment cultures had similar patterns of
response to the various inhibitory stresses with the exception of phosphate. The findings
of this study provide comprehensive insights on the tolerance of the anammox process to
a wide variety of potential inhibiting compounds.
3.2
Introduction
Since anaerobic ammonium oxidation (anammox) was discovered in the early
1990s, it has gained attention due to its importance in the global nitrogen cycle (den
Camp et al., 2006). Anammox is catalyzed by chemolithoautotrophic bacteria (Strous et
al., 1999a) belonging to five genera Brocadia, Kuenenia, Anammoxoglobus, Jettenia and
Scalindua of the phylum Planctomycetes (Harhangi et al., 2011). The anammox reaction
51
involves the oxidation of ammonium (NH4+) coupled to the reduction of nitrite (NO2-) to
produce the main product, N2 and a minor product, nitrate (NO3-) (Eq. 1), under
anaerobic conditions (van der Star et al., 2007; Kuenen, 2008).
NH 4+ + 1.32 NO2− + 0.66HCO3− + 0.13H + →
1.02 N 2 + 0.26 NO3− + 0.066CH 2 O0.5 N 0.15 + 2.03H 2 O
( 4.1)
The postulated reaction mechanism involves the conversion of NH4+ and NO2- to
the intermediate hydrazine (N2H4), which finally leads to the production of N2. Recent
studies propose nitric oxide (NO) as one of the intermediates of the anammox reaction
(Kartal et al., 2011).
Due to ever-increasing stricter nutrient-nitrogen discharge limits, there is a great
need to improve wastewater treatment processes. Compared to the conventional
nitrification-denitrification process, anammox provides a more environmentally
sustainable and cost-effective alternative for nitrogen removal in wastewaters with low
C/N ratio (Renou et al., 2008; Kartal et al., 2010b). However, waste streams commonly
contain compounds that might pose inhibitory effects on anammox activity (AA).
Additionally, anammox bacteria have a slow growth rate with reported doubling times
around 10 to 12 d (Strous et al., 1998; van der Star et al., 2007). Thus, the impact of a
52
severe toxic event, killing the biomass, would be particularly problematic for anammox
due to the long recovery periods needed. Understanding of the factors influencing the
activity of anammox bacteria is essential to improving its applicability, including the
identification of potential toxic compounds present in specific wastewaters.
The objective of this study was to investigate the inhibitory impact of commonly
occurring compounds on two different anammox enrichment cultures dominated by
bacteria from the genus Brocadia. The compounds tested consisted of anammox
substrates (NO2- and NH4+), metabolites (NO3- and N2H4) and constituents frequently
encountered in wastewater (H2S, O2, NaCl, PO43-). Additionally the anammox toxicity of
two commonly utilized nitrification inhibitors was tested. The inhibition was evaluated in
batch assays by comparing the N2 production rates in the absence or presence of each
compound supplied in a range of concentrations.
3.3
3.3.1
Material and methods
Microorganisms
Two enriched anammox cultures (granular and suspended) dominated by
Candidatus Brocadia were used as inoculum. The suspended enrichment culture (SEC)
53
was washed and centrifuged in a NaCl (1%) solution, and re-suspended with basal
medium before transferring into batch assays. This anammox enrichment was originally
developed from a returned activated sludge (Sun et al., 2011) and maintained in a
membrane bioreactor. The granular anammox enrichment (GEC) was obtained from a
full-scale anammox bioreactor operated by Paques BV, Balk, The Netherlands, and
maintained in an expanded granular sludge bed reactor. The volatile suspended solids
(VSS) content of the GEC sludge was 5.69 ±0.04% of the wet weight. The average size
of the Anammox granules was 2.4±0.6 mm. This value was calculated by image analysis
of a photograph of a granular sludge sample using the software ImageJ.
3.3.2
Batch bioassays
Batch assays were performed in serum flasks (160 ml), and incubated on a shaker
(115 rpm) in the dark at 30±2 ˚C. The anammox biomass was added to the assays at 5
vol% for the anammox SEC and 0.6 g VSS L-1 for the GEC. Flasks were supplied with
100 ml basal mineral medium (pH 7.4-7.5) (described in the APPENDIX A), containing
bicarbonate (2.5 g L-1) as the only added carbon. The medium was also supplemented
with a stoichiometric mixture (1.32:1, mol NO2-:mol NH4+) of NO2- (3.57 mM) and NH4+
(2.71 mM), unless otherwise described. Abiotic controls were prepared by excluding the
54
addition of microbial inoculum. Controls lacking NH4+ were included to measure
background endogenous (denitrification) consumption of NO2-.
The flasks were sealed with rubber stoppers, and then the medium and the
headspace were purged with He/CO2 (80/20, v/v) to exclude oxygen from the assays. All
assays were conducted in duplicate. Headspace samples were analyzed periodically for
N2 gas content to monitor the AA. Liquid samples were extracted at the beginning and at
the end of each experiment and analyzed for NO2-, NO3- and NH4+. Experiments testing
the inhibitory effect of phosphate were supplemented with phosphate concentrations
ranging 5-50 mM (supplied as NaH2PO4•H2O/ Na2HPO4•7H2O at a molar ratio of 0.23).
The corresponding controls contained 0.42 mM phosphate.
3.3.3
Assessment of anammox activity and inhibition
The anammox activity (AA) was measured based on the N2 production rate and
expressed as mmol N2-N Lliquid-1 h-1. The AA was calculated from the maximum slope of
the time course of the N2-N concentration in the headspace as follows: AA= ∆N2/∆t
(mmol N2 Lliquid-1 h-1). The only exception was for the inhibitory effect of dissolved
oxygen (DO) and N2H4, which was assessed based on the NO2- consumption rate (mM h1
). The inhibition was expressed as the relative AA (RAA) (%)= [AAinhibitor/AAreference] ×
55
100. In the experiment assessing the effect of different initial pH values, the AA at the
optimal pH was chosen as the AAreference value. The measurements of liquid
concentrations of NO2-, NO3- and NH4+ were used to confirm the anammox reaction.
Consumption of NO2- and NH4+ as well as formation of NO3- (data not shown)
corresponded to the N2 production according to the stoichiometry of anammox reaction in
all batch assays (Eq. 1). The final pH value was measured to confirm that the optimal
range was maintained.
3.3.4
Analytical methods
NO3- and NO2- were analyzed by suppressed conductivity ion chromatography, N2
and O2 by gas cromatography with termal conductivity detector, and NH4+ using a NH4+
ion selective electrode (Sun et al., 2011). Liquid samples for sulfide or hydrazine
determination were collected prior to addition of NH4+ and NO2-. Sulfide was analyzed
spectrophotometrically by the methylene blue method (Truper and Schlegel et al., 1964).
Hydrazine was analyzed according to Van der Star (2008). All other measurements (pH
and VSS) were according to Standard Methods (APHA, 2005). More information about
analytical procedures is provided in the SM section.
56
3.3.5 Molecular methods
Anammox bacteria in SEC and GEC were characterized by generating a clone
library as described in the APPENDIX A.
3.4
3.4.1
Results and discussion
Characterization of enrichment cultures
The two different inocula were characterized by different anammox species. One
unique anammox phylotype was found in each of the enrichment cultures, both showing
very high similarity with the 16S rRNA gene of the genus Brocadia (> 99.5%). The
anammox strain in the SEC was most closely related to Brocadia caroliniensis, whereas
the GEC was enriched in Brocadia fulgida (Fig. A1). The maximum specific AA of the
SEC and GEC cultures was 0.48±0.06 and 0.19±0.02 mmol N2 g-1 VSS d-1, respectively.
The pH optima of these cultures were characterized in assays incubated at pH values
ranging from 6.5 to 8.3 (based on final pH measurements). The highest AA was achieved
at pH 7.5 and 7.3 for SEC and GEC, respectively (Fig. 3.1). Both cultures had sharp pH
optima, and important losses in activity exceeding 20% were evident as the pH shifted
more than 0.3 units from the optimal pH values. These results showed some disagreement
57
with a previous study (Egli et al., 2001) reporting a wider optimal pH range (7.5- 8.0).
However, a recent study indicated a sharp decrease of AA at pH below pH 7.2 (van der
Star et al., 2007). The impact of pH extremes has been rationalized based on the existance
of an energy yielding intracellular proton gradient over the anammoxosome membrane
(van der Star et al., 2010). Long term alteration of the pH of the medium could lead to
disruption of the proton motive force and thus affecting the associated energy generation.
Inhibition has also been attributed to the pH-dependent, unionized and presumably more
toxic forms of the substrates, HNO2 and NH3 (Anthonisen et al., 1976; Fernandez et al.,
2012).
Figure 3.1. Effect of pH on the anammox activity of a granular enrichment
culture (●) and a suspended enrichment culture (○).
58
3.4.2 Effects of anammox substrates and metabolites
To evaluate ammonium toxicity, the enrichment cultures were incubated at
different concentrations of NH4+ (2.7-44.2 mM) while keeping the NO2- concentration
constant at 3.6 mM. The AA slightly decreased with increasing NH4+ concentration (Fig.
3.2a). At the highest NH4+ concentration tested (44 mM), the inhibition observed for
GEC and SEC was only 16 and 34%, respectively. The results demonstrated low levels of
inhibition caused by NH4+, which agrees with previous reports (Strous et al., 1999b;
Dapena-Mora et al., 2007). Free ammonia is believed to be the actual inhibitor of
anammox (Waki et al., 2007; Fernandez et al., 2012; Aktan et al., 2012). In contrast with
ammonium, undissociated ammonia is expected to diffuse over the microbial cell
membrane. At the pH level tested in this study (7.2), the maximum concentration of free
ammonia in the experiments was 6.5 mg L-1. This concentration is significantly lower
compared to inhibitory values reported for free ammonia (50% inhibition at 46 mg L-1)
(Fernandez et al., 2012).
59
Figure 3.2. Effect of anammox reaction substrates and products on the anammox
activity of a granular enrichment culture (■) and a suspended enrichment culture (□)
exposed to: (a) NH4+, (b) NO2-, (c) NO3-, and (d) N2H4.
To examine the inhibitory effect of NO2-, batch assays with different initial NO2concentrations (3.6-53.9 mM) were performed at the same concentration of NH4+ (2.7
mM) (Fig. 3.2b). No significant inhibition was observed for NO2- concentrations lower
than 7.4 mM. However, NO2- concentrations of 15.1 mM or higher dramatically impacted
the AA. At this concentration, no N2 production was detected in assays with SEC, while
60
only 16.4% RAA was observed in the GEC. The IC50 of NO2- was 10.8±0.05 mM and
13.2±0.13 mM for the SEC and GEC, respectively. These results coincided well with
previously reported inhibitory concentrations (Egli et al., 2001; Dapena-Mora et al.,
2007), although in one study 7 mM NO2- was required to completely inhibit an anammox
enrichment culture from a sequencing batch reactor (Strous et al., 1999b).
Several
authors have attributed the inhibitory effect of nitrite on anammox bacteria to the
undissociated species, nitrous acid (HNO2) (Jin et al., 2012, Fernandez et al., 2012).
However, a recent study has demonstrated that NO2- is the actual inhibitor and that
nitrous acid is not responsible for inhibition (Lotti et al., 2012).
The latter study
hypothesized that the presence of ladderane membrane lipids may hinder diffusion of
HNO2, preventing changes in pH that can lead to loss of the proton motive force.
The inhibitory impact of NO3- (5.5-100 mM) was evaluated since as a soluble
product of anammox, it can potentially accumulate. The RAA of the enrichments
decreased by 15-20% (SEC) or 40-50% (GEC) when exposed to NO3- concentrations
ranging from 5.5 to 20.5 mM (Fig. 3.2c). Inhibition was much more severe when the
NO3- concentrations were 50 mM or higher.
N2H4 is an intermediate of the anammox reaction (Schalk et al., 1998; Kartal et
al., 2011). Bioassays were conducted to evaluate the impact of hydrazine (0.02-2.6 mM)
61
on AA. A 40% increase of the NO2--consumption rate was observed for GEC (Fig. 3.2d).
In the case of SEC, no significant stimulation was observed and modest inhibition was
observed at 2.7 mM. Accumulation of ammonium occurred in tests with high
concentration of hydrazine, and final N2 production was higher than that of the control
lacking hydrazine. Addition of N2H4 at concentration of 0.1 mM has been reported to
enhance the full recovery of AA from cultures previously inhibited by NO2- (Strous et al.,
1999b). In our tests, either stimulation or moderate inhibition was observed at
concentrations higher than 0.3 mM. However at the lowest concentration tested (0.03
mM), slight stimulation was observed in both enrichments (RAA of 103 and 113% for
SEC and GEC, respectively). In test supplied with 2.5 mM hydrazine as the sole nitrogen
source, the two cultures had similar hydrazine removal rates, ranging from 1.79 to 1.99
nmol min-1 mg-1 VSS. The similarity in rates despite large differences in specific AA
might be attributed to the different capability to perform the disproportionation of
hydrazine by different anammox bacteria (Schalk et al., 1998).
3.4.3
Effects of common wastewater constituents
Sulfide is commonly found in anaerobic reactors as a product of mineralization of
organic matter or sulfate reduction. The effect of sulfide on AA was investigated in
assays supplied with 0.1-1.0 mM Na2S. The toxicity of sulfide has often been associated
62
with its unionized form (H2S), therefore the results are expressed in terms of
undissociated H2S rather than total sulfide added. The equation used to calculate the
dissolved undissociated concentration based on pH, total S added and headspace to liquid
volume ratio is provided in the SM. Undissociated H2S caused serious inhibition of the
AA with IC50 values of 0.03 and 0.11 mM for the SEC and GEC, respectively (Fig. 3.3).
A concentration of unionized H2S as low as 0.32 mM caused complete inhibition of the
SEC. The GEC was also highly inhibited but it was able to conserve a small RAA
(24±4%) at higher concentrations (0.9 mM). No H2S consumption was observed in either
abiotic or biological treatments during the course of the experiment (data not shown).
H2S has been previously shown to cause complete anammox inhibition at concentrations
of 0.65 mM (Dapena-Mora et al., 2007). In another study, application of pulses of sulfide
of 2 mM caused stimulation of ammonium consumption in a fluidized bed anammox
reactor (van de Graaf et al., 1996). This phenomenon was explained by the reduction of
nitrate by sulfide, producing nitrite, which is the preferred electron acceptor of the
anammox process. Our study shows that anammox biomass is severely inhibited at very
low sulfide concentrations, not previously reported in the literature. The strong effect of
sulfide could be related to the high dependence of anammox process on heme proteins
(Kuenen, 2008). Sulfide has been reported to interact with heme centers of cytochrome
oxidase as well as to cause reduction of the heme iron in cytochrome c (Pietri et al.,
2011), which could potentially lead to disruption of anammox metabolism.
63
Figure 3.3. Effect of H2S on the anammox activity of a granular enrichment
culture (♦) and a suspended enrichment culture (◊).
Anammox is considered to be strongly inhibited by O2 (Strous et al., 1997), yet
there are a number of processes in which DO is present while nitritation and anammox
are simultaneously taking place in the same bioreactor (Strous et al., 1997; Wett, 2007).
To better understand the tolerance of anammox cultures to DO exposure, different
amounts of O2 were added to the anaerobic headspace to achieve DO equilibrium
concentrations ranging 1-8 mg L-1 (0.03-0.25 mM). The two enrichment cultures had
similar inhibitory responses (Fig. 3.4). At low DO of 1 mg L-1 (0.03 mM), only partial
inhibition of less than 20% was observed. However, at ambient saturation of DO (8
mg·L-1), the AA was severely inhibited. The IC50 value determined for DO was 3.8±0.6
64
mg L-1 (0.12±0.02 mM) and 2.3±0.03 mg L-1 (0.07±0.001 mM) for SEC and GEC;
respectively. No nitrification was observed in tests inoculated with the SEC and only
12.5% ammonium was nitrified in GEC controls with 20% O2 in the headspace, fed with
ammonium only (data not shown). Exposure to oxygen has been reported to cause
different levels of anammox inhibition (Jetten et al., 1998; Strous et al., 1998; Egli et al.,
2001). For example, Strous et al., (1998) reported a 90% inhibition on the AA with DO
concentration of 0.25 mM. Egli et al., (2001) reported that exposure to low DO
concentrations (0.25-1% O2 vol. in the gas phase) caused reversible inhibition of AA, but
irreversible inhibition was observed at higher DO concentrations (18% O2). As shown in
this study, considerable AA is observed at relatively high DO concentrations. In fact,
although ammonium consumption was observed in GEC controls fed with just NH4+ in
the presence of 20% O2 in the headspace, NO2- accumulation was not observed; instead
simultaneous N2 gas production was measured. The data suggest that NO2-, slowly
produced by nitrification, was readily utilized by anammox bacteria even at the high DO
concentrations.
65
Figure 3.4. Effect of O2 on the anammox activity of a granular enrichment culture
(●) and a suspended enrichment culture (○).
Phosphate is commonly found in wastewaters due to its use in detergents and
fertilizers (Alamdari and Rohani, 2007). The impact of phosphate at concentrations
ranging from 5.5 to 50 mM on the AA was investigated. The results (data not shown)
demonstrated that SEC exposure to phosphate caused a modest decrease of RAA with
increasing phosphate concentration (IC50 = 25.3±5.9 mM). On the other hand, phosphate
stimulated the RAA of GEC by 60% at concentrations ranging 10-50 mM. Van Graaf et
al., (1996) observed complete inhibition of a suspended anammox enrichment exposed to
5 mM phosphate. Dapena-Mora et al., (2007) reported a higher tolerance of anammox
biofilms from a gas-lift reactor to phosphate (IC50 = 20 mM). These results suggest that
66
the impact of phosphate on AA is highly dependent on the aggregation degree of the
biomass. Anammox biofilms are more tolerant to high phosphate concentrations
compared with suspended biomass.
A variety of industrial effluents have a high salt content, therefore the impact of
salinity on AA is a concern. The SEC was incubated with different NaCl concentrations
(50 - 300 mM). The RAA decreased with increasing NaCl levels, and the calculated IC50
was 93±4 mM. At concentration of 200 mM and higher, the AA was completely
inhibited. The inhibitory behavior demonstrated a linear relationship with NaCl
concentration (R2 = 0.995, data not shown). The genera Candidatus Kuenenia and
Candidatus Scalindua have been shown to be relatively tolerant to high salinity with
observable activity up to 1.54 M in previously adapted cultures (Kartal et al., 2006;
Dapena-Mora et al., 2007). Our results suggest that the genera Brocadia may be less
suitable for treating high salinity effluents.
3.4.4
Effects of nitrification inhibitors
The effect of nitrification inhibitors was evaluated since inhibitors could
potentially be used in studies evaluating contribution of anammox in microaerophilic
environments (Table 1). Compounds such as allylthiourea (ATU), or nitrapyrine are
67
commonly used to supress enzyme ammonium monooxygenase for ammonium oxidation
(Robertson et al., 1989). Chlorate (ClO3-) has been reported to selectively inhibit nitrite
oxidation in soil samples (Belser and Mays, 1980). ATU did not inhibit GEC at 0.034
mM, but caused 40% inhibition of the SEC activity at 0.043 mM. This is in disagreement
with a previous study (Dapena-Mora et al., 2007), which tested much higher ATU
concentrations (8.6 mM) without observing any appreciable effect. Nitrapyrine caused a
slight stimulation of N2 production rate at low concentrations (0.011 - 0.024 mM), but
was inhibitory at concentrations higher than 0.023 mM, with an IC50 of 0.061±0.002 mM.
In contrast to the other nitrification inhibitors, ClO3- caused a severe and irreversible
inhibition on AA of SEC with an IC50 of 0.04±0.002 mM. This is the first report of ClO3inhibition to anammox.
68
Table 3.1. Effects of various common wastewater contaminants and nitrification inhibitors
on the activity of a suspended- and a granular anammox enrichment culture
Tested
concentrations
(mM)
Contaminants
Nitrification
inhibitors
Suspended
culture
Granular
culture
NH4+
2.99-44.15
GMC*
GMC
NO2-
4.18-53.92
10.76±0.05
13.22±0.13
NO3-
5.5-100
32.00±0.39
21.23±1.22
N2H4
0.02-2.65
GMC
GMC
Phosphate
0.42-100
25.29±5.88
GMC
O2
0-0.625
0.12±0.02
0.071±0.001
Salinity as
NaCl
0-300
92.69±4.26
N/Aa
H2S
0.028-3.19
0.03±0.001
0.096±0.014
Allylthiourea&
0-0.09.
0.04±0.004
GMC
Nitrapyrin&
0-21
N/A
14.17±0.51
ClO3-
0-0.6
0.04±0.002
N/A
Substrates and
products
Wastewater
Constituents
IC50
(mM)
&
The molecular weights of allylthiourea and nitrapyrin are 116.19 g·mol-1 and 230.91 g·mol-1,
respectively.
* GMC, greater than the maximum concentration tested.
a
N/A means not available.
69
3.5
Implications
The anammox process is a promising tool for the treatment of wastewaters with
high NH4+ and and low C content. Due to the slow growth of anammox bacteria,
exposure of the microorganisms to inhibitory compounds should be avoided in order to
ensure a fast start-up and guarantee stable process operation.
Two different inocula, a granular and a SEC characterized by two different
anammox strains, were exposed to various toxicants. Both cultures showed similar levels
of inhibition by each toxicant applied, with exception of phosphate that caused
stimulation of the granular sludge and inhibited the activity of the suspended culture at
concentrations from 20 to 100 mM. Among the wastewater constituents studied in this
work, H2S and DO are of major importance. Sulfide is generated by decay of biomass
and sulfate reduction in anaerobic environments, and it has been shown to cause complete
inhibition of the anammox reaction at concentrations as low as 0.3 mM unionized H2S
(Fig. 3.3). In effluents where sulfide is present, measures should be taken to remove it
prior to anammox treatment, e.g. by addition of iron (III) to precipitate sulfide. Although
anammox microorganisms are inhibited in the presence of DO, considerable AA was
observed at relatively high DO concentrations, which makes the combination of the
70
anammox process with partial nitritation feasible to accomplish complete nitrogen
removal in a single reactor.
The AA is very dependent on the pH level. A sharp decrease of AA occured when
the pH level shifted 0.3 pH units from the optimum, thus pH control is a critical
parameter for the operation of the anammox process.
71
CHAPTER 4
PRE-EXPOSURE TO NITRITE IN THE ABSENCE OF AMMONIUM STRONGLY
INHIBITS ANAMMOX
4.1
Abstract
Anaerobic ammonium oxidizing bacteria (Anammox) are known to be inhibited
by their substrate, nitrite. However, the mechanism of inhibition and the physiological
conditions under which nitrite impacts the performance of anammox bioreactors are still
unknown. This study investigates the role of pre-exposing anammox bacteria to nitrite
alone on their subsequent activity and metabolism after ammonium has been added.
Batch experiments were carried out with anammox granular biofilm pre-exposed to
nitrite over a range of concentrations and durations in the absence of ammonium. The
effect of pre-exposure to nitrite alone compared to nitrite simultaneously fed with
ammonium was evaluated by measuring the anammox activity and the accumulation of
72
the intermediate, nitric oxide. The results show that the inhibitory effect was more
dramatic when bacteria were pre-exposed to nitrite in absence of ammonium, as revealed
by the lower activity and the higher accumulation of nitric oxide. The nitrite
concentration causing 50% inhibition was 53 and 384 mg N L-1 in the absence or the
presence of ammonium, respectively. The nitrite inhibition was thus 7.2-fold more severe
in the absence of ammonium. Biomass exposure to nitrite (25 mg N L-1), in absence of
ammonium, led to accumulation of nitric oxide. On the other hand when the biomass was
exposed to nitrite in presence of ammonium, accumulation of nitric oxide was only
observed at much higher nitrite concentrations (500 mg N L-1). The inhibitory effect of
nitrite in the absence of ammonium was very rapid. With 74% loss in activity during the
first 30 min. The results taken as a whole suggest that nitrite inhibition is more acute
when anammox cells are not actively metabolizing. Accumulation of nitric oxide in the
headspace most likely indicates disruption of the anammox biochemistry by nitrite
inhibition, caused by an interruption of the hydrazine synthesis step.
4.2
Introduction
The anaerobic oxidation of ammonium (NH4+) (anammox) is a novel technology
for the removal of nitrogen pollution from wastewaters. The anammox process is
catalyzed by chemolithoautotrophic bacteria of the phylum Planctomycetes that use
73
nitrite (NO2-) as terminal electron acceptor and NH4+ as an electron donor, allowing for
NH4+ removal in the absence of oxygen (Strous et al., 1999a). Anammox is advantageous
over the traditional nitrification-denitrification process for nutrient-N containing effluents
since oxygen needs are decreased by up to 57%, and no additional electron donor is
needed as would otherwise be the case for denitrification. Unlike other prokaryotes,
anammox bacteria have a complex internal compartmentalization. The central organelle,
called anammoxosome is the locus of the anammox metabolism (Kartal et al., 2011). The
catabolism of anammox bacteria involves the reduction of NO2- to nitric oxide (NO) by a
nitrite oxidoreductase (NirS). Subsequently, hydrazine synthase enzyme (HZS) forms
hydrazine (N2H4) by combining NH4+ with NO. Lastly, N2H4 is oxidized to dinitrogen
gas (N2) by hydrazine dehydrogenase (HDH) (Kartal et al., 2011). The oxidation of N2H4
produces four high energy electrons that are used to generate an intracellular proton
gradient which energizes the production of ATP (van der Star et al., 2010).
Inhibition of anammox microorganisms by substrates and intermediates has been
extensively studied. NH4+ has been found to cause low inhibition corresponding to a 50%
inhibiting concentration (IC50) of 770 mg NH4+-N L-1 (Dapena-Mora et al., 2007).
Similarly, the intermediates NO and N2H4 cause little or no inhibition to anammox
(Carvajal-Arroyo et al., 2013a, Schalk et al., 1998). On the other hand, different levels of
anammox inhibition by NO2- have been reported in batch and continuous reactors. Strous
et al., (1999b) found complete inhibition of the anammox activity at NO2- concentration
74
of 100 mg N L-1, while other authors have reported higher tolerance to NO2- with IC50
values of 350 mg N L-1 (Dapena-Mora et al., 2007) and 400 mg N L-1 (Lotti et al., 2012).
Decreases in nitrogen removal efficiency, due to NO2- overload in a full scale anammox
reactor, have also been reported (van der Star et al., 2007). The undissociated species,
free nitrous acid (FNA), has been suggested to be responsible for the inhibitory effect of
NO2- to anammox bacteria (Fernandez et al., 2012, Jaroszynski et al., 2011). On the other
hand other researchers claim that the inhibition is only dependent on the total NO2concentration (Lotti et al., 2012).
NO2- is known to cause toxicity in a wide variety of microorganisms (Philips et
al., 2002). FNA, acting as a protonophore, inhibits the production of adenosine
triphosphate (ATP) by disrupting bacterial transmembrane proton gradients (Sijbesma et
al., 1996). Inhibition of different enzymes by NO2- has been reported (He et al., 2006,
Titov and Petrenko 2003). In some cases NO2- radicals or reactive derivatives are
responsible for the toxicity (Hurst and Lymar 1997). The reactive nitrogen species can
bind to biomolecules such as the well-known formation of nitrotyrosine from reaction
with tyrosine moieties (Monzani et al., 2004).
The mechanisms controlling the inhibitory impact of NO2- on anammox bacteria
and the conditions under which NO2- impacts the performance of the anammox process
75
are still unclear. Therefore, control of NO2- inhibition remains a difficulty in the
application of anammox reactors. In this work, the impact of pre-exposing anammox
bacteria in granular biofilms to NO2- alone was compared with exposure to NO2- during
active metabolism (when NO2- is added simultaneously with NH4+). Moreover the
potential generation of toxic by-products during NO2- exposure was evaluated. The
inhibitory effect of NO2- was evaluated in batch assays by comparison of the anammox
activity and accumulation of the intermediate NO in anammox cultures previously preexposed to NO2- in the presence or absence of NH4+.
4.3
4.3.1
Materials and Methods
Origin of the biomass
All the experiments were inoculated with anammox granular sludge cultivated
and maintained in a laboratory-scale expanded granular sludge bed (3 L) fed with a
synthetic medium at a loading rate of 3.7 g N L-1 d-1. The reactor was originally
inoculated with anammox granular sludge provided by Paques BV (Balk, The
Netherlands) from a full-scale anammox wastewater treatment plant in The Netherlands.
This inoculum was used to start up the reactor which was operated for one year before
carrying out the experiments. The volatile suspended solids (VSS) content of the biomass
76
from the laboratory reactor was 5.69 ±0.04% of the wet weight. The average size of the
anammox granules was 2.4±0.6 mm (calculated by image analysis of a photograph of the
granular sludge sample using the software ImageJ). Bacteria from the genus Brocadia
were the dominant anammox microorganisms in the sludge granules (Carvajal-Arroyo et
al., 2013a).
4.3.2 Batch bioassays
Batch assays were performed in duplicate and incubated on an orbital shaker (160
rpm) in the dark at 30±2˚C. Serum flasks (160 mL) were supplied with basal mineral
medium (100 mL) and anammox biomass (0.71 g VSS L-1). The mineral medium was
prepared using ultrapure water (Milli-Q system; Millipore) and contained the following
compounds (mg L-1): NaH2PO4· H2O (57.5), CaCl2·2H2O (100), MgSO4·7H2O (200), and
1.0 mL L-1 of two trace element solutions. Trace element solution 1 contained (in mg L1
): FeSO4 (5,000), and ethylenediamine-tetraacetic acid (EDTA) (5,000). Trace element
solution 2 contained (in mg L-1): EDTA (1,500), ZnSO4·7H2O (430), CoCl2·6H2O (240),
MnCl2
(629),
CuSO4·5H2O
(250),
Na2MoO4·2H2O
(220),
NiCl2·6H20
(190),
Na2SeO4·10H2O (210), H3BO3 (14), and NaWO4·2H2O (50). Either NaHCO3 (47.6 mM),
phosphate (30 mM) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (25
mM) were utilized as buffer systems, as described below. The serum flasks were sealed
77
with rubber stoppers and aluminum crimp seals. When NaHCO3 buffer was used, the
liquid and the headspace were purged with a gaseous mixture of He/CO2 (80/20, v/v),
resulting in a final pH of 7.1-7.2. In the case of using HEPES buffer, the medium was
supplemented with NaHCO3 (50 mg L-1) as carbon source, and the pH was adjusted to
7.2 with NaOH. Liquid and the headspace was purged with ultra-high purity He. When
phosphate buffer was used, NaH2PO4 and Na2HPO4 were added at a molar ratio of 0.46
and the medium was also supplemented with NaHCO3 (50 mg L-1). The pH in these
experiments was 7.3.
Table 4.1 summarizes the test conditions utilized in the various experiments. The
addition of NO2- and NH4+ to the bioassays was performed following the protocols
described below and depicted in Fig. 4.1.
Protocol 1: NH4+ and NO2- were added simultaneously (“simultaneous
exposure”). The experiments were carried out in a mineral medium with NaHCO3/CO2 as
buffer system.
Protocol 2: Bioassays were supplemented with NO2- and pre-incubated for
different time periods ranging up to 24 h (“NO2--pre-exposure”). After the pre-exposure
period, bioassays were spiked with NH4+. In treatments where the residual concentration
78
of NO2- was lower than 50 mg N L-1, NO2- was supplemented to attain 50 mg N L-1. The
experiments were carried out in a mineral medium with NaHCO3/CO2 as a buffering
system.
Protocol 3: The biomass was pre-exposed to NO2- for 24 h. After that period, the
biomass was allowed to settle and the liquid was decanted and replaced by 100 mL of
mineral medium containing no N-compounds. This process was repeated twice to ensure
that no NO2- remained in the granules (“washed granules”). Subsequently, the bottles
were closed and flushed with He, and NH4+ and NO2- were added simultaneously. In
order to avoid pH variations during the manipulation of the flasks, these experiments
were carried out in HEPES buffer and in phosphate buffer.
In assays performed according to protocols 2 and 3, controls were included where
the biomass was pre-exposed to NH4+ (76 mg N L-1) (in absence of NO2-) during the
“pre-exposure period.” The controls were supplemented with NO2- (100 mg N L-1) after
24 h of incubation. Likewise, controls were included in which no N-containing substrates
were added during the pre-exposure period. These controls were supplemented with NO2and NH4+ after 24 h of incubation.
79
Protocol 4: In these experiments, fresh anammox biomass was incubated with
anaerobically decanted liquid medium obtained from a nitrite pre-exposure assay.
Afterwards the medium was supplemented with NH4+, the bottles were sealed and purged
with He. The experiments were carried out in HEPES- or in phosphate-buffered medium.
Table 4.1. Summary of conditions applied to each experiment.
Pre-exposure Period
Monitoring Period
Experiment
Protocol
NO2(mg N L-1)
Time
(h)
NO2(mg N L-1)
NH4+
(mg N L-1)
NO2-inhibition
in presence of
NH4+
Protocol 1
-
-
50-500
38
NO2-inhibition
in absence of
NH4+
Protocol 2
0-100
24
50-100
38
Effect of the
length of the
pre-exposure
period
Protocol 2
100
0-12
100
76
Washing Effect
Protocol 3
100
24
100
76
Toxicity of preincubated
medium
Protocol 4
100
24
100
76
80
In all the cases, samples of the headspace were analyzed for N2 and NO at the
beginning and at the end of the pre-exposure period, and periodically, after addition of
NH4+. Liquid was sampled after addition of the substrates and at the end of the
experiments, for analysis of NH4+, NO2- and NO3-.
Figure 4.1. Experimental protocols for addition of substrates in batch bioassays.
81
4.3.3 Analytical methods
Nitrate (NO3-) and NO2- were analyzed by suppressed conductivity ion
chromatography using a Dionex IC-3000 system (Sunnyvale, CA, USA) fitted with a
Dionex IonPac AS18 analytical column (4 × 250 mm) and an AG18 guard column (4 ×
50 mm). During each run, the eluent (15 mM KOH) was used for 20 min. NH4+ was
determined using a Mettler Toledo SevenMulti ion selective meter with a Mettler Toledo
selective NH4+ electrode (Mettler Toledo, Columbus, OH, USA). N2 was analyzed using
a Hewlett Packard 5890 Series II gas chromatograph (Agilent Technologies, Palo Alto,
CA, USA) fitted with a Carboxen 1010 Plot column (30 m x 0.32 mm) and a thermal
conductivity detector. The temperatures of the column, the injector port and the detector
were 220, 110 and 100°C, respectively. Helium was used as the carrier gas and the
injection volume was 100 µL. NO was analyzed using a chemiluminescence detector
model NOA 280i (General Electric, Fairfield, CT, USA). The VSS content was analyzed
according to Standard Methods (APHA, 2005).
4.3.4
Assessment of specific anammox activity and inhibition
The specific anammox activity (SAA) was measured based on the N2 production
rate and expressed as g N g VSS-1 d-1. The SAA was calculated from the maximum slope
82
of the time course of the N2 concentration in the headspace as follows: (SAA) = ΔN2 (g
VSS Δt)-1. The activity of each experiment was normalized with respect to the activity of
a control not subjected to inhibitory conditions, normalized anammox activity (nAA, %)
= (SAAinhibited/SAAcontrol) x 100. The concentration of NO2- causing 50% inhibition (IC50)
was calculated by interpolation in the graphs plotting the nAA as a function of the NO2concentration.
4.4
4.4.1
Results
Inhibition by NO2- in the presence or absence of NH4+
Since NO2- is a substrate and inhibitor of anammox bacteria, its impact on the
anammox process may be different depending on whether it is being actively metabolized
or not. Thus the presence of NH4+ may affect the tolerance of anammox bacteria to NO2inhibition. The effect of NO2- on the nAA of anammox granular sludge was evaluated
over a range of NO2- concentrations in the presence of NH4+ and compared to the residual
nAA of biomass pre-exposed to NO2- in absence of NH4+ during 24 h. The inhibitory
effect of NO2- was greatly enhanced in the absence of NH4+. Fig. 4.2A compares the time
course of N2 formation with and without pre-exposure to NO2- (100 mg N L-1). In
experiments where NH4+ and NO2- were fed simultaneously from the beginning, rapid
83
production of N2 occurred. However, in experiments where the biomass was first preexposed to NO2- for 24 h prior to the NH4+ addition, there was essentially no N2
production for 22 h after subsequent addition of NH4+. Fig. 4.2B shows the nAA as a
function of NO2- concentration with and without NO2- pre-exposure for 24 h. The graph
clearly illustrates there is large difference in the impact of NO2- depending on whether it
was pre-exposed or fed simultaneously with NH4+. The IC50 values were 53 and 384 mg
NO2--N L-1 for incubations pre-exposed to NO2- and simultaneous incubations,
respectively. Complete inhibition was observed at 100 and 500 NO2--N L-1, respectively.
Based on the IC50 values, the NO2- was approximately 7.2-fold more inhibitory when preexposed compared to simultaneous feeding. Controls pre-exposed to NH4+ (instead of
NO2-) caused no detrimental effect nor did starving the anammox biomass of both NO2and NH4+ have any negative impact (Fig. B1; APPENDIX B). Thus it is the exposure to
NO2- alone rather than a short starvation period that was responsible for the impact.
84
A
Production N2 (mg N L-1)
100
80
60
40
20
0
5
10
15
20
Time (h)
25
30
B
100
nAA (%)
75
50
25
0
100
200
300
400
Concentration NO2 (mg N L-1)
500
Figure 4.2. Impact of NO2- pre-exposure on NO2- inhibition. A, Time course of N2
production with NO2- pre-exposure for 24 h ( ) and with simultaneous NO2- and NH4+
feeding ( ). The NO2- and NH4+ concentrations used were 100 and 38 mg N L-1,
respectively. B, The effect of NO2- concentration on the nSAA after pre-exposure for 24 h
to NO2- alone ( ) or when simultaneously fed with NH4+ and NO2- ( ). The maximum
SAA in simultaneously fed treatments and pre-exposed treatments, was 0.33±0.02 and
0.40±0.02 g N g VSS-1 d-1, respectively
85
4.4.2 Effect of the duration of the pre-exposure period
A separate experiment was designed in order to determine how rapidly the NO2- preexposure inflicts full impact. Anammox granular sludge was pre-exposed to NO2- (100
mg N-NO2- L-1) for different periods of time prior to the addition of NH4+ in order to test
the role of pre-exposure time on inhibition of anammox metabolism (Table 4.1, Figure 3).
The results obtained show that anammox inactivation by NO2- occurred quickly. After
only 30 min of pre-exposure to NO2- the inhibition was 74%. When the pre-exposure was
continued up to 12 h, the observed inhibition approached 100%. (Fig 4.3). Thereafter,
SAA (mg N mg VSS-1 d-1)
further increments in the inhibition required more time to impart further losses in activity.
1.0
0.8
0.6
0.4
0.2
0.0
0
2
4
6
8
10
Pre-exposure time (h)
12
Figure 4.3. Effect of the time of pre-exposure to 100 mg NO2--N L-1 on nAA of
anammox sludge in absence of NH4+. The SAA of the non-pre-exposed control was
0.98±0.02 g N g VSS-1 d-1.
86
4.4.3 Role of the liquid medium pre-incubated with NO2-
The strong inhibition observed following exposure of anammox bacteria to NO2- preexposure (Fig. 4.2) could be due to the formation of toxic byproducts during preincubation. To test this hypothesis, anammox bacteria were exposed to decanted culture
media obtained from bioassays pre-exposed to NO2- for 24 h. The procedure followed in
these assays was according to protocol 4 (Fig. 4.1). Likewise, the medium of anammox
bacteria pre-exposed to NO2- for 24 h was decanted; the biomass was washed and the
assays were replaced with fresh medium to determine if the washing reversed the toxicity
or if the anammox cells were damaged by the NO2- pre-exposure. The experimental
procedure followed in these assays was protocol 3 (Fig. 4.1).
Fig. 4.4 compares the nAA of biomass simultaneously fed with NH4+ and NO2(A), biomass pre-exposed to NO2- (B), biomass washed after pre-exposure to NO2- (C),
and healthy biomass exposed to a medium decanted from a 24 h NO2- pre-exposure
treatment (D). These bioassays were conducted both in HEPES and phosphate buffer.
Washing the biomass after the pre-exposure period caused a moderate relief in the
inhibition. The observed nAA after recovery was higher in HEPES buffer (42%) than in
phosphate buffer (20%). The use of a medium pre-incubated with NO2- caused inhibition
of healthy biomass (22% in HEPES buffer, and 39% in phosphate buffer). These results
87
indicate that inhibition occurred partly due to the inactivation of the biomass by NO2- and
partly due to the formation of soluble toxic intermediates.
Figure 4.4. The nAA of simultaneously fed biomass (A), biomass pre-exposed
to NO2- (B), biomass washed after biomass pre-exposure (C), and healthy
biomass exposed to a medium decanted from another NO2- pre-exposed assay
(D). Bars indicate the buffer system used in each experiment: HEPES buffer
( ), phosphate buffer ( ). The SAA of the simultaneous fed controls (A) were
0.80±0.03 and 0.92±0.04 g N g VSS-1 d-1 in HEPES and phosphate buffer,
respectively.
88
4.4.4 Nitric oxide accumulation
Accumulation of NO gas, an anammox intermediate (Kartal et al., 2011), was observed in
the headspace of bioassays inhibited by exposure to NO2-. In experiments where the
biomass was exposed simultaneously to NO2- and NH4+ , accumulation of NO was only
observed in treatments with very high NO2- levels (500 mg NO2--N L-1) coinciding with
conditions in which N2 production was completely inhibited (Fig. 4.5). In contrast, NO
gas was detected at much lower NO2- concentrations if the biomass was pre-exposed to
NO2- (Fig. 4.6). Such conditions occurred when the NO2- concentration was equal or
higher than 25 mg NO2--N L-1. As shown in Fig. 4.6, the concentration of NO in the
headspace of these assays increased with the increasing concentrations of NO2- in the preexposure period. At 500 mg NO2--N L-1, the 24 h production of NO in simultaneously fed
cultures (Fig. 4.5) was the same as that of 100 NO2--N L-1 in NO2- pre-exposed cultures
(Fig. 4.5) suggesting that a 5-fold lower NO2- concentration was needed to achieve the
same impact on NO production in the pre-exposed cultures.
89
Figure 4.5. - Time course of N2 production (closed symbols) and NO
accumulation (open symbols) at initial NO2- concentrations of 57 (diamonds),
and 500 (squares) mg NO2--N L-1 when a simultaneous feeding protocol of NO2and NH4+ was utilized.
The impact of the duration of NO2- pre-exposure (100 mg NO2--N L-1) on
anammox activity and NO accumulation was also evaluated (Fig. 4.7). The increasing
durations of the pre-exposure had increasing inhibitory impacts on the anammox activity
and caused parallel increases in the NO accumulation. By comparison, NO levels in the
control were below the detection limit. At the end of the pre-exposure period, the levels
of NO in the headspace were considerably higher in treatments exposed to NO2- for
longer periods. After addition of NH4+ (76 mg N L-1), the concentration of NO in the gas
90
phase continued to increase with time in all the pre-exposed treatments suggesting that
the anammox bacterial cells were damaged. However, NO levels decreased gradually
after 2 to 6 h of incubation after NH4+ addition, which occurred in synchrony with the
moment there was partial recovery in the anammox activity.
Figure 4.6. NO gas accumulation after 24 h of pre-exposing the anammox
biomass to different concentrations of NO2- in the absence of NH4+.
91
Figure 4.7. Time course of N2 (A) and NO (B) produced after pre-exposing
the anammox biomass to 100 mg NO2--N L-1 for 0 min (●), 15 min (♦), 1h
(▲) and 12 h (■)
92
4.5
4.5.1
Discussion
Influence of physiological state on NO2- inhibition of anammox bacteria
The results obtained indicate that the inhibitory effect of NO2- to anammox is
greatly enhanced in the absence of NH4+ (Fig. 4.2). The IC50 value determined for NO2was 7.2 times lower when the anammox culture was pre-exposed to NO2- (nonmetabolizing) versus simultaneous exposure to NH4+ that enables active metabolism.
Although a NO2- concentration of 100 mg N L-1 has been reported to be a safe limit for
operation of anammox bioreactors (Jin et al., 2012), the results in this work show that
under non-metabolizing conditions, considerable inhibition can occur following preexposure to lower NO2- concentrations.
Non-metabolic pre-exposure to NO2- was shown to cause detrimental impact very
quickly, with 74% of the full impact occurring within 30 min of incubation (Fig. 4.3).
The results in Fig. 4.3 suggest that in the absence of NH4+ the initial toxic effect is occurs
immediately as NO2- contacts the cells. After 1 h of pre-exposure, further contact with the
NO2- does not cause a proportional decrease in activity. This could be due to two reasons.
The first may be an active mechanism of detoxification carried out by cells. The second
may be due to additional time required to extensively damage biomolecules. Other
93
authors have suggested that mass transfer limitation may impact the extent of the
inhibition caused by NO2-. Suspended biomass has been shown to be more sensitive to
NO2- than granular sludge (Cho et al., 2010), and the highest tolerance to NO2- inhibition,
reported in the literature, was observed in anammox biomass embedded in a gel carrier
(Kimura et al., 2010).
In order to determine if the toxicity caused by pre-exposure of anammox cells to
NO2- is irreversible, the cells were washed prior to addition of NH4+ and NO2-. The
washing caused a modest recovery of the SAA, when compared to the non-washed
biomass (Fig. 4.4). This indicates that the NO2- toxicity is only partially reversible by
washing, confirming that a large portion of the toxicity observed was due to a lasting
damage to cells. The low recovery of activity after biomass washing obtained in this
work contrasts with the high recovery rates previously reported in the literature. For
example, Scaglione (2012) observed almost complete recovery of SAA after preexposure to 100 mg NO2--N L-1 for 48 h, and Lotti (2012) observed that the SAA
decrease after exposure to 500 mg N L-1 was not higher than 35%. In these two cases the
NO2- concentration applied after the washing was lowered to 50 mg N L-1, while in our
case the level of NO2- was the same as in the pre-exposure period (100 mg N L-1). Other
factors such as pH differences may have also contributed to the divergence. The medium
pH is known to have a marked influence on NO2- inhibition to anammox activity
(Jaroszynski et al., 2011). The medium pH used in the current study was 7.2-7.3; whereas
94
the previous studies applying washing used pH values ranging from 7.5-7.7 (Lotti et al.,
2012, Scaglione et al., 2012).
Additionally, activity tests performed with liquid medium recovered from the preincubation with NO2- (100 mg N L-1) showed that this medium inhibited healthy
anammox cells (38.8 and 21.5% in phosphate and HEPES buffers, respectively) (Fig.
4.4). These findings indicate that a toxic by-product may have been formed during the
pre-incubation period. The difference in the results obtained in HEPES buffer and
phosphate buffer may be related to the chemistry of the formation of the toxic byproduct, favored in phosphate buffered medium.
The formation of toxic NO2- derivatives in biological medium has been previously
reported (Philips et al., 2002). NO and intermediates produced by NO2- reduction can
potentially generate other toxic products like nitrogen dioxide or peroxynitrite anion with
high reactivity against biomolecules, including DNA, lipids, or proteins (e.g.,tyrosine
residues) (Mehl et al., 1999). The reaction products of NO2- and different aminoacids
were shown to be more toxic to activated sludge, than NO2- itself (Philips et al., 2002).
4.5.2
NO accumulation, evidence of disrupted anammox cells
NO gas accumulated in experiments where NO2- inhibition occurred. Five times more
NO2- were needed to cause NO gas accumulation in experiments with simultaneous
95
exposure than in NO2- pre-exposed treatments. The amount of NO accumulated in the
headspace depended on the NO2- concentration as well as on the length of the preexposure period (Figs. 4.6 and 4.7B).
In anaerobic environments, NO can be generated chemically or, from biological
reduction of NO2- by denitrification or anammox. NO can be generated chemically from
the reaction of Fe2+ in the medium and NO2- (Kampschreur et al., 2011). The observed
dependence of the NO production on the NO2- concentration could suggest a chemical
reaction as the source of the NO in our assays. Nevertheless, chemical formation of NO
was discarded from evidence in abiotic controls containing 100 mg NO2--N L-1, in which
NO could not be detected. Another possible source of NO gas is endogenous
denitrification. Experiments where the granular sludge was amended with NO2- (100 mg
N L-1), and hydrogen or methanol at stoichiometric concentrations, were monitored for
longer than 24 h, and denitrifying activity could not be detected (Fig. B2; APPENDIX
B). Therefore, anammox seems to be the source of the NO. The low amount of NO
detected is consistent with the use of an endogenous source of electrons by anammox
bacteria. As shown in Fig. 4.7, the accumulation of NO did not cause a complete halt in
the N2 production, and therefore the accumulation of NO seems to be an indicator of
disruption of anammox metabolism by NO2- inhibition, rather than the cause of the
inhibition (Kartal et al., 2010b). NO and NH4+ are the substrates of the enzyme hydrazine
synthase which produces N2H4, later oxidized to N2. The accumulation of NO under
96
conditions of NO2- inhibition suggests that this step of the anammox catabolism may be
interrupted.
4.5.3
Hypothesis for NO2- inhibition
The absence of NH4+ enhances the toxic effect of NO2-. The big difference in the extent
of the inhibition observed under metabolizing conditions or under non-metabolizing
conditions (in absence of NH4+) suggests that the inhibition does not depend only on the
NO2- concentration but also in the physiological status of the cells.
Three different phenomena could explain this behavior. Firstly, active anammox
metabolism is only made feasible in the presence of NH4+, which provides a sink for
NO2-, lowering its concentration to non-toxic levels. Clearly the accumulation of NO2- in
the anammoxosome is due in part to this first phenomena since without NH4+ there will
be no metabolism of NO2-. Consequently it will accumulate and potentially inhibit HZS
and as a consequence NO will accumulate as was witnessed in this study. The NO
accumulation and anammox inhibition linger even after adding NH4+.
Secondly, mechanisms of NO2- detoxification are probably dependent upon the
availability of metabolic energy to pump NO2- out of sensitive regions of the cell. If cells
97
are non-metabolizing, NO2--pumps will not be active. Consequently NO2- may not be
adequately pumped out of sensitive areas of the cell (e.g. riboplasm, anammoxosome)
where lasting damage to biomolecules can potentially be imparted. Anammox catabolism
leads to the generation of an intracellular proton gradient between both sides of the
anammoxosome membrane (van der Star et al., 2010). NO2- active transport proteins
(NirC) have been found in the anammox genome (van de Vossenberg et al., 2013), which
are H+ and NO2- symporters dependent on a transmembrane proton motive force.
Therefore, the capability of anammox bacteria to actively metabolize NH4+ and NO2- ,
and maintain the proton gradient, will directly affect the active transport of NO2- between
the anammoxosome and the other compartments (e.g. riboplasm). Some authors have
suggested that the intracellular proton gradient is positive inside the anammoxosome (van
der Star et al., 2010, van Niftrik and Jetten 2012), therefore NirC could be involved in
NO2- detoxification, translocating NO2-. In order to validate this mechanism, the role of
NO2- transport proteins in anammox bacteria, as well as the effect of the pH on NO2inhibition need to be further investigated.
Thirdly NH4+ may act as a reductant for the proper turn-over of enzymes and their
cofactors. Inactivated oxidized enzyme cofactor requires electron equivalents to properly
turn over. There is ample evidence that NO2- inhibited cells can be rapidly recovered
using highly reduced substrates such N2H4 or hydroxyl amine (NH2OH) (Bettazzi et al.,
2010, Strous et al., 1999b).
98
The higher permeability of biological membranes to undissociated compounds has led
to the belief that free nitrous acid and not the NO2- anion causes inhibition of anammox
bacteria (Fernandez et al., 2012, Jaroszynski et al., 2011). Nevertheless, the uniqueness of
the ladderane anammoxosome membrane (Fuerst et al., 2006) has been suggested to be a
barrier for FNA passage (Lotti et al., 2012), and therefore the mechanism of NO2accumulation in the anammoxosome would not depend only on the bulk concentration of
free nitrous acid.
4.6
Implications
The anammox process can be inhibited by nitrite. NO2- inhibition is not only
dependent on the bulk NO2- concentration, but also on the physiological status of the
cells. The susceptibility of anammox bacteria to inhibition by NO2- is higher when NH4+
is not available. On the other hand, when NH4+ is actively being metabolized, anammox
bacteria have a higher resistance to NO2- inhibition. The inhibitory effect of NO2- in
absence of NH4+ occurs very quickly, impacting the activity of the cells in a matter of
minutes. The detrimental effect of NO2- can be partially reverted by washing of the cells.
The anammox process is applied in combination with a previous step, of partial
nitrification, were approximately half of the NH4+ is oxidized to NO2-. This can be done
in different configurations (i.e., CANON, SHARON, sequencing batch reactor). This
99
work shows that the operation of the nitritation step is critical for the safe application of
the anammox process. An event resulting in complete oxidation of NH4+ to NO2- , during
the nitritation step, could lead to failure of the anammox process. Or if NH4+ and NO2are being pumped into an anammox reactor from two different sources, a failure of the
NH4+ delivery pump could have a serious inhibitory impact. Strategies must be followed
to avoid such events and, in the case that they occur, measures need to be in place to
minimize the duration of the disturbance.
100
CHAPTER 5
THE ROLE OF pH ON THE RESISTANCE OF RESTING- AND ACTIVE
ANAMMOX BACTERIA TO NO2- INHIBITION
5.1
Abstract
The anaerobic oxidation of ammonium (anammox) uses nitrite as terminal
electron acceptor. The nitrite can cause inhibition to the bacteria that catalyze the
anammox reaction. Currently there is no consensus on whether free nitrous acid or
ionized nitrite is responsible for the toxic effect. This work investigated the effect of the
pH and the concentration of nitrite on the activity and metabolism of anammox granular
sludge under different physiological conditions. Batch activity tests in a range of pH
values were carried out in which either actively metabolizing cells or resting cells were
exposed to nitrite in the presence or absence of the electron donating substrate
ammonium, respectively. The response of the bacteria was evaluated by analyzing the
101
specific anammox activity, the accumulation of nitric oxide, and the evolution of the ATP
content in the biomass. Additionally, the effect of the pH on the tolerance of the biomass
to single substrate feeding interruptions was evaluated in continuous anammox
bioreactors. The results show that the concentration of free nitrous acid alone cannot be
used to predict the inhibition of actively metabolizing anammox bacteria. At pH higher
than 7, the ionized NO2- concentration is more predictive of the inhibition. The exposure
of resting cells to NO2- (100 mg N L-1) at pH values below 7.2 caused complete inhibition
of the anammox activity. The inhibition was accompanied by accumulation of the
intermediate, nitric oxide, in the gas phase. In contrast, just mild inhibition was observed
for resting cells exposed to the same NO2- concentration at pH values higher than 7.5 or
any of the pH values tested in assays with actively metabolizing cells. ATP initially
increased and subsequently decreased in time after resting cells were exposed to NO2suggesting an active response of the cells to nitrite stress. Furthermore, bioreactors
operated at pH lower than 6.8 had greater sensitivity to NO2- during an ammonium feed
interruption than a bioreactor operated at pH 7.1. The results suggest that actively
metabolizing biomass is resistant to nitrite toxicity over a wide range of pH values;
whereas the ability of resting cells to tolerate NO2- inhibition is seriously impeded at
mildly acidic pH values.
102
5.2
Introduction
Anaerobic ammonium oxidation (anammox) is novel technology for biological
nitrogen removal in NH4+-rich, carbon-poor wastewaters. The anammox reaction is
catalyzed by chemolithoautotrophic bacteria of the phylum Planctomycetes that use
ammonium (NH4+) as electron donor and nitrite (NO2-) as terminal electron acceptor
(Strous et al., 1999a). The anammox process is advantageous over traditional
nitrification-denitrification process since it allows for significant savings in aeration,
electron donor used for denitrification is not needed, and, due to their low cell yield, the
sludge production is low (Strous et al., 1999a). Anammox cells are divided in different
compartments separated by lipidic membranes. The metabolism of anammox bacteria
occurs in the anammoxosome (Kartal et al., 2011), the central organelle that is
surrounded by a unique ladderane lipid membrane (Fuerst et al., 2006). In this organelle,
NO2- is first reduced to nitric oxide (NO) which is later combined with NH4+ yielding
hydrazine (N2H4). Finally, N2H4 is oxidized to dinitrogen gas (N2). Upon N2H4 oxidation,
a pH gradient is generated over the anammoxosome membrane that fuels the production
of ATP (van der Star et al., 2010).
Among the compounds involved in the anammox reaction, NO2- has gained
attention due to its potential to cause inhibition of anammox bacteria. Evidence of NO2-
103
inhibition can be found in various literature reports. In batch experiments, complete
inhibition at 100 mg NO2--N L-1 was reported by Strous (1999), while other authors
observed 50% inhibition at nitrite concentrations ranging from 350 to 400 mg NO2--N L-1
(Dapena-Mora et al., 2007, Lotti et al., 2012). Similarly to the results obtained with
nitrifying bacteria (Anthonisen et al., 1976), NO2- inhibition of anammox bacteria has
frequently been attributed to the undissociated species, free nitrous acid, HNO2 (FNA)
(Fernandez et al., 2012). On the other hand, some authors concluded that inhibitory
impact of nitrite was caused by the NO2- anion (Lotti et al., 2012). Anammox activity has
been found in a wide pH range (7-9) (Egli et al., 2001) with the optimum at pH 7.2-7.4
(Carvajal-Arroyo et al., 2013a). The pH impacts directly the speciation of HNO2/NO2-,
but it can also affect anammox bacteria by altering other metabolic processes which rely
on the pH gradients, e.g., energy generation by ATPases or pH dependent active transport
proteins, some of which are NO2- transporters (Lu et al., 2013, van de Vossenberg et al.,
2013). Some studies have reported enhanced inhibition of anammox bacteria when NO2was supplied alone, in a pre-exposure period, prior to addition of NH4+ (Carvajal-Arroyo
et al., 2013b, Scaglione et al., 2012), suggesting that factors other than
the FNA
concentration may be implied in the mechanism of NO2- inhibition.
The conditions affecting the tolerance of anammox bacteria to NO2- inhibition are
not well understood. There is no consensus on the contribution of the unionized FNA and
the dissociated NO2- to the inhibition of the anammox bacteria. In this work, batch and
104
continuous experiments were carried out to study the influence of the pH on the response
of anammox granular sludge when pre-exposed to NO2- alone (resting cells) and when
simultaneously supplied with NH4+ (metabolically active cells). The effect of NO2- was
evaluated by comparing the anammox activity, the accumulation of the intermediate NO
gas, and the ATP content of the sludge under different pH conditions.
5.3
5.3.1
Materials and Methods
Origin of the biomass
Anammox granular sludge was used in all the experiments. The sludge was
cultivated and maintained in a 3-L laboratory-scale expanded granular sludge bed
(EGSB) reactor fed with synthetic medium at a loading rate of 3.7 g N L-1 d-1. The reactor
was originally inoculated with anammox granular sludge provided by Paques BV (Balk,
The Netherlands) from a full-scale anammox wastewater treatment plant in The
Netherlands. The average size of the granules was 2.4±0.6 mm (calculated by image
analysis using the software ImageJ). The volatile suspended solids (VSS) content of the
biomass from the nursing reactor was 5.69±0.04% of the wet weight.
105
5.3.2
Batch bioassays
Batch activity tests were performed in duplicate and incubated in an orbital shaker
(160 rpm) in a dark climate controlled room at 30±2 ºC. The serum flasks (160 mL) were
supplied with basal mineral medium (100 mL) and inoculated with 0.71 g VSS L-1 of
anammox granules. The mineral medium was prepared using ultrapure water (Milli-Q
system; Millipore, Billerica, MA, USA) according to the following recipe (mg L-1):
NaH2PO4•H2O (57.5), CaCl2•2H2O (100), MgSO4•7H2O (200), and 1.0 mL L-1 of two
trace element solutions. Trace element solution 1 contained (in mg L-1): FeSO4 (5,000),
and ethylenediamine-tetraacetic acid (EDTA) (5,000). Trace element solution 2 contained
(in mg L-1): EDTA (1,500), ZnSO4•7H2O (430), CoCl2•6H2O (240), MnCl2 (629),
CuSO4•5H2O (250), Na2MoO4•2H2O (220), NiCl2•6H2O (190), Na2SeO4•10H2O (210),
H3BO3 (14), and NaWO4•2H2O (50). Either NaHCO3, or 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) were utilized as buffer systems, as described
below. The serum flasks were sealed with rubber stoppers and aluminum crimp seals. In
bicarbonate buffered experiments, the amount of NaHCO3 added (21.2-190.5 mM) was
selected to obtain the target pH (6.8-7.8) after purging the liquid and the headspace with a
gaseous mixture of He/CO2 (80/20, v/v). For high pH experiments (8.0-8.2) the liquid
and headspace were flushed with ultrahigh purity He. In the case of using HEPES buffer,
the medium was supplemented with NaHCO3 (50 mg L-1) as carbon source, and the pH
106
was adjusted to 7.2 with a concentrated solution of NaOH. Liquid and the headspace
were purged with ultra-high purity He.
The substrates were added by injection of concentrated solutions of NaNO2 and
NH4HCO3. In pre-exposure experiments, the bottles were supplemented with either NO2or NH4+, and incubated for a “pre-exposure period” of 24 h prior to addition of the
missing substrate. In simultaneous exposure experiments, both substrates were fed
together to the concentration desired in each experiment.
5.3.3
Continuous bioreactors
Three laboratory-scale UASB reactors (500 mL) were operated in parallel. Each
reactor was inoculated with 1.24 g VSS L-1 of anammox granular sludge and incubated in
a dark climate controlled room at 30±2 ºC. The reactors were fed with a basal mineral
medium (described above), at a hydraulic retention time of 0.25 d. The medium was
supplemented with NH4+ and NO2- at 108 and 129 mg N L-1, respectively. For operation
at pH values of 6.5 and 7.2, the concentration of NaHCO3 was 0.5 and 2.5 g L-1
respectively, and the medium was flushed with He/CO2 (80/20, v/v). During operation at
pH 8.3, the medium was supplemented with NaHCO3 (2.5 g L-1) and flushed with
ultrahigh purity He.
107
The three reactors were operated during 8 days at pH 7.2. Subsequently, the pH of
the reactors R1 and R3 was manipulated as described in Table 5.1. In order to study the
effect of substrate accumulation in the reactors, the feeding of NH4+ or NO2- to the
reactors was interrupted on day 18 and 32 for periods of 48 h.
Table 5.1. pH of the influent of the anammox bioreactors during the
different operation periods.
Operation period
Reactor
Period I
Period II
Period III
0-8 d
9-40 d
40-end
7.2
6.5
7.2
R1
7.2
7.2
7.2
R2
7.2
8.3
R3
The N2 production was measured by liquid displacement using a 2% (w/v) NaOH
solution to scrub the CO2 out of the biogas. The performance of the reactors was
monitored by measuring the N2 generation, as well as the pH value and NH4+, NO2- and
NO3- concentration in the influent and effluent.
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5.3.4
ATP extraction and quantification
The granules (0.10-0.12 g wet weight) were disintegrated with a syringe and
suspended in 9 mL of boiling Tris-EDTA (20-2 mM) buffer. The closed vials were
incubated in boiling water for 2 min and then submerged in an iced-water bath. The ATP
in the extract was quantified according to the manufacturer’s instructions using a
commercial ATP determination kit (Life Technologies, Grand Island, NY, USA).
Bioluminescence was analyzed in a fluorescence spectrometer (Model LS-55, Perkin
Elmer, USA), see APPENDIX C. The VSS content of the wet granules in each sample
was quantified.
5.3.5
Analytical methods
Nitrate (NO3-) and NO2- were analyzed by suppressed conductivity ion
chromatography using a Dionex IC-3000 system (Dionex, USA) fitted with a Dionex
IonPac AS18 analytical column (4 × 250 mm) and an AG18 guard column (4 × 50 mm).
During each run, the eluent (15 mM KOH) was used for 20 min. The flowrate was 1 mL
min-1. NH4+ was determined using a Mettler Toledo SevenMulti ion selective meter with
a Mettler Toledo selective NH4+ electrode (Mettler Toledo, USA). N2 was analyzed using
a Hewlett Packard 5890 Series II gas chromatograph (Agilent Technologies, USA) fitted
109
with a Carboxen 1010 Plot column (30 m x 0.32 mm) and a thermal conductivity
detector. The temperatures of the column, the injector port and the detector were 220, 110
and 100°C, respectively. Helium was used as the carrier gas and the injection volume was
100 µL. NO was analyzed using a chemiluminescence detector model NOA 280i
(General Electric, USA). The VSS content was analyzed according to Standard Methods
(APHA 2005).
5.3.6
Assessment of specific anammox activity and inhibition
The specific anammox activity (SAA) was measured based on the N2 production
rate and expressed as g N g VSS-1 d-1. The SAA was calculated from the maximum slope
of the time course of the N2 concentration in the headspace as follows: (SAA) = ∆N2 (g
VSS ∆t)-1. The anammox activity in each assay was normalized with respect to the
activity of a control not subjected to inhibitory conditions, normalized anammox activity
(nAA, %) = (SAAinhibited/SAAcontrol) x 100. The concentration of NO2- causing 50%
inhibition (IC50) was calculated by interpolation in the graphs plotting the nAA as a
function of the NO2- concentration.
110
5.4
5.4.1
Results
The role of FNA on the inhibition of actively metabolizing anammox bacteria
NO2- inhibition of the anammox process has been commonly attributed to FNA
due to its higher ability to diffuse through cell membranes (Anthonisen et al., 1976). A
set of batch experiments was carried out to explore the effect of NO2- and FNA on the
activity of actively metabolizing anammox bacteria simultaneously fed NH4+ and NO2-.
Different concentrations of FNA were applied by alternating combinations of total NO2concentrations (60-800 mg N L-1) and pH values (6.6-7.8) in HEPES buffered medium.
Figure 5.1A shows that both total NO2- and pH affected the SAA. The maximum SAA
was observed at a NO2- concentration of 160 mg N L-1 but higher concentrations caused a
linear decrease in the activity of the biomass. The inhibition did not occur
instantaneously. Inhibited treatments showed a gradual decrease of the SAA during the
first 2.4 h (Fig. C1; APPENDIX C). The pH also influenced the inhibitory effect of NO2-.
When inhibitory levels of NO2- were applied, lower IC50 values were observed at lower
pH, e.g. the IC50 at pH 7.4 was 621 mg NO2--N L-1, whereas at pH 7 the IC50 was 442 mg
NO2--N L-1. The SAA was decreased the most with a combination of high NO2concentration and low pH values, which are conditions expected to correspond to the
highest FNA levels. Although these findings could potentially be interpreted to mean that
111
FNA is correlated to anammox inhibition, Figure 5.1B shows that the FNA
concentrations are not predictive of the SAA. Very disparate SAA values were observed
at equal concentrations of FNA, e.g. no inhibition was observed when FNA was 0.038
mg N L-1 at a pH value of 7, but 71% decrease in activity was observed at 0.039 mg N L-1
when the pH was 7.8. These results show that although in the lower range of pH tested
(6.6-7.0) the SAA is well described by the concentration of FNA, at higher pH values, the
FNA grossly overestimates the inhibition of the anammox activity.
112
SAA (g N g VSS-1 d-1)
A
0.8
0.6
0.4
0.2
0.0
0
200
400
600
800
SAA (g N g VSS-1 d-1)
Concentration Total NO2- (mg N L-1)
0.8
B
0.6
0.4
0.2
0.0
0.0
0.1
0.2
0.3
Concentration FNA (mg N L-1)
Figure 5.1. Effect of Total NO2- (Panel A) and FNA (Panel B) on the anammox activity
at pH = 6.6 ( ), pH = 7 ( ). pH = 7.3-7.4 (∆) and pH = 7.7-7.8 () in metabolically
active cells fed NH4+ and NO2- simultaneously.
113
5.4.2
The role of the pH on the inhibition by NO2- pre-exposure (resting cells)
The inhibitory effect of NO2- is greatly enhanced when cells are metabolically
non-active (resting cells) as observed in experiments where anammox bacteria were preexposed to NO2- only, prior to the addition of NH4+ (Carvajal-Arroyo et al., 2013b,
Scaglione et al., 2012). In the present study, the effect of pre-exposing the granules to
NO2- (100 mg N L-1) was evaluated at different pH values. After the pre-exposure period,
NH4+ (76 mg N L-1) was added, and the pH of each treatment was kept constant. For
comparison, the effect of the pH was also evaluated with metabolically active cells where
NH4+ and NO2- were supplied simultaneously. The NO2- inhibition of resting cells was
seriously impacted by the pH. When the medium pH was near 7.1, 100 mg N L-1 of NO2caused complete inhibition of anammox resting cells (Fig. 5.2). In contrast, no inhibition
occurred if NO2- and NH4+ were fed simultaneously to metabolically active cells at a
similar pH. Likewise, no NO2--inhibition was observed at pH 7.5, irrespective of whether
resting or active cells conditions were used during NO2- exposure.
114
Figure 5.2. Time course of N2 production by metabolically active
anammox cells simultaneously exposed to NH4+ (76 mg N L-1) and NO2- (100
mg N L-1) at pH 7.03 (▲) and 7.52 ( ), and NO2- pre-exposed resting cells
(100 mg N L-1) subsequently supplemented with NH4+ (76 mg N L-1) at pH
7.11 ( ) and 7.52 ( ).
A series of assays comparing exposure of resting and metabolically active cells to
NO2- (100 mg N L-1) were conducted with over a complete range of pH values. The SAA
is plotted as a function of pH for two different buffering systems (NaHCO3/CO2 and
HEPES) (Fig. 5.3A). Assays inoculated with resting cells pre-exposed to NO2- were
highly inhibited at pH values lower than 7.2. The activity gradually increased with pH,
approaching the values in assays simultaneously fed with NH4+ when the pH increased up
to the range of 7.5-7.6. This behavior was in stark contrast to the simultaneously fed
115
treatments which had a high SAA at all pH values including the pH range of 6.8 to 7.2
which was highly inhibitory for the NO2--pre-exposed resting cells. The SAA of the
simultaneously fed treatments incubated in bicarbonate buffer started to decrease at pH
values higher than 7.4, but this was due to the high concentration of NaHCO3 required
(>10 g NaHCO3 L-1) to maintain mildly alkaline conditions with 20% CO2 in the flush
gas; thereby imposing salt inhibition (Carvajal-Arroyo et al., 2013a). Assays performed
in HEPES buffer, on the other hand, provided high SAA activities up to the highest pH
value tested of 8.2.
The concentration of NO was measured in the head space of all the experiments.
A high concentration of NO gas, up to 1820 ppmv accumulated during the pre-exposure
period in the resting cell treatments with pH values lower than 7.4 (Fig. 5.3B). In most
treatments, NO accumulation stopped after addition of NH4+ with the exception of the
treatment with the highest NO level, which continued accumulating NO although at a
much lower rate than during the pre-exposure period. Similarly to the SAA, the
accumulation of NO resting cell treatments was influenced by the pH. The highest NO
concentrations were detected in treatments under pH 7.2, which also showed the highest
levels of inhibition. Much lower accumulation of NO was observed as the pH approached
7.4 and higher, corresponding to conditions with the highest SAA activity after the NO2pre-exposure.
116
SAA (g N g VSS-1 d-1)
1.0
A
0.8
0.6
0.4
0.2
0.0
Accumulation NO (ppmv)
6.8
7
7.2 7.4 7.6 7.8
pH
8
1800
8.2
B
1200
600
0
7.0
7.2
7.4
pH
7.6
7.8
Figure 5.3. Effect of the pH on the response of anammox granular sludge to NO2exposure. A, SAA of metabolically active biomass simultaneously exposed to NO2(100 mg N L-1) and NH4+ (76 mg N L-1) (closed symbols) and SAA of resting cells preexposed for 24h to NO2- (100 mg N L-1) subsequently supplemented with NH4+ (76 mg
N L-1) in bicarbonate buffer (squares) and HEPES buffer (circles). B, Accumulation of
117
NO gas in the headspace after 24h of pre-exposure to NO2- only (100 mg N L-1) at
different pHs, with HEPES buffer (circles) and bicarbonate buffer (squares). Transient
accumulation of trace NO was also observed during incubation of metabolically active
cells simultaneously fed NO2- and NH4+ (data not shown).
Trace NO concentrations were also detectable in metabolically active cells,
although the maximum NO concentrations observed were two orders of magnitude lower
than in resting cells. Instead of accumulation, transitory peaks of NO were observed at
pH 7.2 (15 ppmv) and pH 7.0 (27 ppmv), which disappeared after 3 h and 6 h,
respectively (data not shown).
Inhibition of anammox bacteria by nitrite may be related to the availability of
metabolic energy to the anammox cells, since the inhibition is exasperated when the
energy providing NH4+-substrate is absent in the lower range of pH values suitable for
metabolism. Additional experiments were performed to test this hypothesis.
5.4.3
ATP response to NO2- stress
The alteration of the metabolism of anammox bacteria due to nitrite- or pH
inhibition was studied by measuring the evolution of the ATP content of the biomass in
batch tests. In these assays, the bacteria were pre-exposed to 50 mg NO2--N L-1 under
118
different pH values (6.6-8.4). For comparison, pre-exposure to NH4+ (38 mg N L-1) in
absence of NO2- was also evaluated. In both cases, after 24 h of pre-exposure, the
stoichiometric concentration of the missing substrate was supplied to all the treatments,
and N2 gas production was subsequently monitored.
Figure 5.4 shows the evolution of the ATP content of the biomass together with
the time course of N2 production for each treatment. A temporary increase in the ATP
content was observed in treatments with NO2- pre-exposure having low to circumneutral
pH values of 6.6 and 7.1. The ATP content of the biomass peaked after 3.5 h of NO2exposure and subsequently decreased reaching minimum values at the end of the preexposure period. The highest ATP peak was measured in the treatments with the pH of
7.1, which showed a maximum SAA of 0.32±0.04 g N g VSS-1 d-1. Complete inhibition
was caused by exposure to NO2- at the lowest pH value of 6.6. Although in both
treatments the ATP peaked upon addition of NO2-, at pH 7.1, the ATP content remained
at values similar to the initial, whereas in the treatment at pH 6.6, the ATP content
remained low after addition of the NH4+.
119
Figure 5.4. Time course of N2 production (close symbols) and evolution of
the ATP content (open symbols) of resting cells pre-exposed to 50 mg NO2-N L-1(A) and 38 mg NH4+-N L-1 (B) at pH 6.5 (squares), 7.2 (circles) and 8.3
(triangles). The dotted vertical line represents the addition of the missing
substrate, in stoichiometric concentration.
120
In contrast, NH4+ pre-exposure did not cause an increase in the ATP content of
the biomass nor inhibited the anammox bacteria. All the treatments at the highest pH
tested (ranging from 8.5-8.6) showed a moderate reduction in the activity, more likely
caused by exposure to high pH values rather than nitrite inhibition (Jaroszynski et al.,
2011). The results from the batch experiments clearly indicate that exposure of resting
cells to NO2- alone at slightly acidic pH conditions can potentially be disruptive to the
anammox process. Thus such an event should have important consequences during the
continuous operation of anammox bioreactors.
5.4.4 Long term effect of pH on the stability of anammox bioreactors
Three continuous lab-scale UASB reactors with anammox granular sludge were
utilized to evaluate the role of the pH both during stable operation and during events of
substrate interruption. The reactors were operated for 8 days at the same pH (7.2) prior to
switching the pH of the influent (Table 5.1). At this point the N2 production (Fig. 5.5A)
as well as the removal of NO2- and NH4+ (Fig. 5.6) was similar in the three reactors. After
changing the pH of the influent of the reactors R1 and R3 to 6.4-6.8 and 8.1-8.6,
respectively, the N2 production of the reactor exposed to high pH values (R3), started to
decrease gradually, and approached zero after 8 days. The failure of the reactor could be
confirmed by accumulation of NH4+ and NO2- in the effluent as shown in Figure 5.6. In
121
contrast, applying a low pH to R1 did not affect its N removal capacity, and the reactor
showed a N2 production similar to R2. On day 19 and 20, the reactors were fed with a
medium containing just NH4+ as the only substrate by interrupting the supply of NO2-. As
a consequence, NH4+ accumulated in both reactors R1 and R2 (Fig. 5.6A), concomitantly
with the disappearance of the N2 production. The 2-day period of exposure to NH4+ alone
however did not affect the N removal potential of the biomass, in either of the reactors,
which both recovered full treatment capacity after restoring NO2- in the feeding.
122
Figure 5.5. Effect of the pH on the performance of UASB reactors subjected
to NO2- and NH4+ substrate interruption, respectively. Evolution of the daily
N2 production (A) and effluent pH (B) of the reactors R1 ( ), R2( ),
R3( ) during different operation periods (I: start up, II: application of
different pH to each reactor, III: recovery of optimum pH in R1).
123
Forcing exposure of the biomass to NO2- alone by discontinuing of NH4+ feed
between the days 32-34 caused the N2 production to stop and NO2- accumulated up to
128±1 mg N L-1 and 119±12 mg N L-1 in the effluents of R1 and R2, respectively. These
concentrations were approximately the same as those fed to the reactors via the influent.
As opposed to sole exposure to NH4+, the 2-day period of sole exposure to NO2- caused
irreversible failure of R1, operated at pH values ranging from 6.4-6.8 (Fig. 5.5). Upon
reestablishment of NH4+ in the feeding, both substrates accumulated in the effluent of R1,
and no signs of recovery could be observed. Even after the pH of the R1 influent was
increased to 7.1 on day 40, the N2 production remained non-detectable, confirming
complete disruption of the N removal capacity of the biomass. In contrast R2, which had
been operated at pH ranging 7.0-7.4, showed complete removal of NH4+ and NO2- after
NH4+ was supplemented again in the feeding. This immediate recovery of the substrate
consumption rate was not immediately reflected in the N2 production, which showed a
progressive recovery. Nonetheless full N2 production was restored after 3-4 days.
124
Figure 5.6. Concentration of NH4+ (A) and NO2- (B) in the influent (close
symbols) and effluent (open symbols) of the reactors R1 (diamonds), R2
(squares) and R3 (circles), during different operation periods (I: start up, II:
application of different pH to each reactor, III: recovery of optimum pH in
R1).
125
5.5
5.5.1
Discussion
Short term effect of NO2- inhibition
The results taken as a whole indicate that FNA alone cannot be used to predict
inhibition of anammox activity. As shown in Figure 5.1B, equal concentrations of FNA
lead to very different levels of inhibition. On the other hand the inhibitory effect of the
NO2- was influenced by the pH and higher levels of inhibition were observed at lower pH
values (Fig. 5.1A). Other authors have suggested that FNA is responsible for the
inhibitory impact of nitrite toward anammox bacteria (Fernandez et al., 2012) on the
basis of findings derived from studies with nitrifying and denitrifying bacteria
(Anthonisen et al., 1976), but the study did not provide direct evidence to support that
hypothesis. The high variability in the response observed under equal concentrations of
FNA suggests that the ionized form NO2- and the pH affect anammox bacteria
independently of the FNA concentration.
Nitrite inhibition is exacerbated when the resting cells are pre-exposed to NO2prior to addition of NH4+ (Carvajal-Arroyo et al., 2013b). The ability of the resting cells
exposed to the same NO2- to perform the anammox reaction upon subsequent addition of
NH4+ was very much affected by the pH (Fig. 5.2A). Complete inhibition was observed
126
when the pre-exposure period took place at pH below 7.2. Some other studies have
reported enhanced inhibition caused by pre-exposure to NO2- in absence of NH4+ (Lotti et
al., 2012, Scaglione et al., 2012). In these studies the recovery of the activity was
generally high (80% and 75%, at 100 and 250 mg NO2--N L-1, respectively). This can be
explained by the relatively high pH applied during the experiments, ranging from 7.5 to
7.8, and a washing of the cells after the pre-exposure which helped in the recovery of the
activity. As shown by our results, the pH plays a critical role in the damage caused during
pre-exposure of resting cells to NO2-, and maximum activities occur at pH values over
7.5. Lotti et al., (2012) reported that the level of inhibition during NO2- pre-exposure
events was not affected by pH values from 6.8-7.8 which seemingly contradicts the
findings presented here. Nevertheless, their experiments are not really comparable since
their protocol provided a thorough washing of the biomass after the pre-exposure period.
Furthermore, both NO2- concentration and pH were readjusted to non-inhibitory values
(pH = 7.5 and NO2- concentration of 50 mg N L-1), which allows for evaluating the
reversibility of the inhibition rather than analyzing the in situ effect of the cells at the
original pH in the medium remaining after pre-exposure.
The inhibitory effect of NO2- to resting cells has been shown to occur rapidly,
almost in parallel with NO2- diffusion through the granules (Carvajal-Arroyo et al.,
2013b). In contrast, when NO2- is fed together with NH4+, the inhibition was observed to
127
occur progressively, as the N2 production rate decreased during the first 2.4 h, until a
steady value for each treatment was reached (Fig. C1).
The occurrence of NO2- inhibitory conditions resulted in the accumulation of NO
gas (Fig. 5.2B). Aside from anammox bacteria, NO gas could also have been formed by
chemical reaction of NO2- and Fe2+ (Kampschreur et al., 2011). In the present study,
however, chemical production of NO was not detected in abiotic controls. Moreover,
NH4+ greatly influenced NO accumulation which would not be expected if NO generation
of NO had a chemical origin. NO could also be formed by denitrification or nitrifier
denitrification (Kampschreur et al., 2008), but the generation of NO gas was not impacted
by supplying H2 or methanol as electron donors (results not shown). The absence of NO
accumulation in the presence of NH4+ further supports the generation of NO by anammox
bacteria. The accumulation of NO gas, generated by anammox bacteria under conditions
of NO2- inhibition, suggests an interruption of the metabolic steps following NO2reduction, i.e., synthesis of N2H4, and/or generation of N2 gas (Kartal et al., 2011).
The anammox bacteria obtain energy from the transformation of NH4+ and NO2into N2 gas. Therefore, when the anammox reaction is not taking place, a decrease in the
ATP content of the biomass could be expected. In contrast, when the resting cells were
exposed to NO2- at pH 6.6 and 7.1, the ATP levels temporarily increased by 1.6 and 1.8
128
fold, respectively. This response was not observed at high pH or in resting cells preexposed to NH4+. As indicated before the greatest inhibition of resting cells pre-exposed
NO2- occurred at pH lower than 7.2. These results taken together indicate that the ATP
peaks are a response of the cells to NO2- stress, which is more evident in the lower range
of pH tested. In response to a potentially harmful situation caused by excessive NO2-, the
bacteria may be recruiting energy from food storage for detoxification or for repair of cell
damage. As depicted in Figure 5.4A, the ATP content of the biomass pre-exposed to
NO2- at pH 7.1 remained high after the pre-exposure period, whereas in the inhibited
biomass the ATP content became depleted even after addition of NH4+. This suggests that
at low pH values the bacteria need to invest more energy to overcome damage caused by
NO2-. A similar response to NO2- stress was observed in denitrifying poly-phosphate
accumulating bacteria (DPAOs) (Zhou et al., 2010). In DPAOs, the application of NO2caused an increase in phosphorus release and in consumption of glycogen, concomitantly
with transient increase in the cellular ATP levels. Also in Desulfovibrio vulgaris, NO2stress triggered a series of transcriptional responses, including up-regulation of genes
favoring ATP generation by substrate level phosphorylation (He et al., 2006).
129
5.5.2
Mode of action of NO2- and FNA
NO2- is known to cause toxicity on a wide variety of microorganisms. It has a
high affinity for metals in the center of enzymes, and it is very reactive against
biomolecules (Philips et al., 2002), causing nitration of moieties in proteins such as the
production of nitrotyrosine (Monzani et al., 2004). In some cases it has been found that
the reaction products of NO2- (Reactive Nitrogen Species) are even more toxic than
nitrite itself (Philips et al., 2002). Furthermore, FNA is hydrophobic and can pass through
membranes via passive diffusion (Almeida et al., 1995). Due to this property it has been
suggested that FNA could act as a protonophore causing inhibition by disrupting transmembrane proton gradients in various microorganisms, e.g. denitrifying bacteria
(Sijbesma et al., 1996) or even compartmentalized organisms like yeasts (Mortensen et
al., 2008).
We have hypothesized that NO2- inhibition of anammox bacteria occurs due to
accumulation of NO2- in a sensitive area of the cells (e.g., riboplasm, anammoxosome)
(Carvajal-Arroyo et al., 2013b). Accumulation of NO2- can occur through three different
mechanisms: i) low NO2- turnover capability in absence of electron donor, ii) inactivation
of oxidized enzymes due to the lack of reducing equivalents, and iii) interruption of NO2detoxification by active pumps dependent on energy originating from trans-membrane
130
proton motive force. In absence of NH4+, the NO2- is not actively consumed and,
therefore, the ability of the cells to avoid inhibition must be controlled by their capability
to pump NO2- out of the sensitive region. Indeed NO2- transport proteins have been found
in anammox bacteria (NirC) (van de Vossenberg et al., 2013). This enzyme has been
related to detoxification functions in E. coli (Lu et al., 2013). Its primary function would
be facilitated passive NO2- import but, due to the toxicity of NO2-, it also functions as a
facultative secondary active NO2- /H+ exporter, keeping low (non-toxic) intracytoplasmic NO2- concentrations (Jia et al., 2009). The secondary transporter capability
of the enzyme is therefore dependent on the existence of a trans-membrane proton
gradient. Thus, environmental conditions reducing the proton motive force will
necessarily hinder the NO2- export capacity. Interestingly, the presence of toluene was
shown to enhance NO2- inhibitory effect on anammox bacteria (Hernández et al., 2013),
and its role augmenting the permeability of cell membranes was suggested to increase the
toxic effect of NO2-.
As demonstrated by our results, the ability of resting cells to tolerate NO2- preexposure was very dependent on the pH. Even in the presence of NH4+, the inhibition was
stronger when lower pH values were applied. The activation of the NO2- export system
by active pumps works at the expense of the proton motive force. While the anammox
reaction takes place, the proton motive force is actively maintained (van der Star et al.,
2010). Therefore, when the anammox reaction does not occur (e.g. when NH4+ is absent),
131
low pH may potentially dissipate residual proton gradients created by endogenous
metabolism and, as a consequence, an alternative energy source is needed. Anammox
bacteria are known to accumulate glycogen as molecule for energy storage (van Niftrik et
al., 2008a). Therefore, glycogen could be used by the cells as source of energy in cases
when the anammox reaction is not taking place. This could be enough to overcome mild
NO2- toxic events, but insufficient when very harsh conditions are imposed.
5.5.3
The effect of NO2- and pH during continuous operation
The pH is a critical parameter for the operation of anammox reactors. Both the
application of low and high pH values showed advantages and disadvantages. Under low
pH conditions, R1 showed a much higher sensitivity to NO2- inhibition when that
coincided with a 2 d interruption of NH4+ in the feed and the activity of the biomass
could not be recovered afterwards. Just prior to the NH4+-feed interruption, operation of
anammox at pH 6.4-6.8 was suitable for the anammox conversion. The same feed
interruption had a very limited impact on the performance of R2 which was operated at
pH 7.1. The results indicate that a combination of low pH and a feed interruption of NH4+
can have grave consequences for anammox bioreactors. Operation of the bioreactors at
high pH also caused instability in the anammox bioreactor due to high pH or free
ammonia associated with high pH (Jaroszynski et al., 2011).
132
5.6
Implications
NO2- is an inhibitor of anammox bacteria. The inhibitory effect cannot be
predicted solely by the concentration of FNA, because ionized NO2- may play a role in
the observed inhibition, especially when the pH conditions are higher than 7. The
sensitivity of the bacteria to NO2- inhibition strongly depends on pH, and on the
physiological status of the cells. Resting cells are severely inhibited when exposed to
NO2- at mildly acidic pH values, but they tolerate exposure to NO2- when the pH ranges
from 7.4 to 7.8. On the other hand, application of pH values above 8 for extended periods
of time result in inhibition and ultimate inactivation of the biomass. This information
needs to be taken into account during design and operation of anammox bioreactors. In
order to avoid failure of the process, the operation must be kept within a fairly narrow
window with its optimum near 7.4.
133
CHAPTER 6
STARVED ANAMMOX CELLS ARE LESS TOLERANT TO NO2- INHIBITION
6.1
Abstract
Anaerobic ammonium oxidating (anammox) bacteria can be inhibited by their
terminal electron acceptor, nitrite. Serious inhibition of the anammox bacteria by nitrite
occurs if the exposure coincides with the absence of the electron donating substrate,
ammonium, or mildly acidic conditions. Little is known about the effect of the nitrogen
loading rate on the sensitivity of anammox bacteria to nitrite inhibition. Starvation of the
biomass may occur during severe underloading in bioreactors or storage of the biomass.
This work investigated the effect of starvation on the sensitivity of anammox bacteria to
nitrite exposure. Batch activity tests were carried out where anammox biomass subjected
to different levels of starvation was exposed to nitrite in the presence and absence of
ammonium. The response of the bacteria was evaluated by measuring the specific
anammox activity and the evolution of the ATP content in the biomass over time. The
134
effect of starvation on the tolerance of anammox bacteria to nitrite was further evaluated
in continuous bioreactors, by imposing nitrite accumulation (by interrupting the
ammonium feeding), after operation at different nitrogen loading rates. The results show
that starvation impairs the capacity of anammox cells to tolerate nitrite. The 50%
inhibitory concentrations of nitrite in starved- and fresh- resting cells was 7 mg N L-1 and
52 mg N L-1, respectively. Starvation only moderately affected the inhibition caused to
active cells, exposed to nitrite and ammonium simultaneously. The ATP content in
resting cells increased upon addition of NO2-. The maximum ATP content observed in
starved cells was 30% lower than in fresh cells. Moreover, underloading anammox
bioreactors decreased their tolerance to nitrite exposure. Accumulation of 107 mg NO2- N L-1 after operation at 0.95 g N L-1 d-1 did not cause observable inhibition of the
bacteria. On the other hand, relatively similar nitrite levels (101 mg NO2--N L-1)
completely disrupted the N removal capacity of the biomass when the reactor was
underloaded (0.10 g N L-1 d-1).
6.2
Introduction
The anammox process is the microbial catalyzed oxidation of ammonium (NH4+)
using nitrite (NO2-) as electron acceptor, generating N2 gas as major final product. After
its discovery in the early 1990s, the anammox processt has been applied to the treatment
135
of NH4+ rich wastewaters. Due to the chemolithoautotrophic nature of the process and
that elemental oxygen (O2) is not needed, anammox technology is advantageous over
conventional on nitrification – denitrification systems, which are costly and energy
intensive. Anammox cells have a complex internal organization, with three lipid
membranes that divide the cell in several compartments. The central organelle, called
anammoxosome, houses the enzymes responsible for the anammox catabolism (Kartal et
al., 2011). As a result of their catabolism, anammox bacteria generate a transmembrane
proton gradient between both sides of the anammoxosome membrane, which is used for
synthesis of ATP (van der Star et al., 2010). Furthermore the anammox bacteria
accumulate glycogen, a polymer for energy storage that the anammox bacteria may use
for cell maintenance during periods of starvation (van Niftrik et al., 2008).
One of the most intriguing aspects of anammox bacteria is their potential to be
inhibited by one of their substrates, NO2-. Although the literature reporting NO2inhibition of anammox bacteria is abundant, the mechanism by which it occurs in not
known and there is divergence on the threshold levels of NO2- that cause inhibition
(Table 6.1). The physiological status of the cells may affect the resistance of anammox
bacteria to NO2- (Lotti et al., 2012, Scaglione et al., 2012 and Chapter 4), being resting
cells more sensitive to NO2- than metabolically active cells (Chapter 4). Furthermore, the
toxic effect caused by NO2- , is enhanced when the pH is low (Chapter 5).
136
Table 6.1. Reported data about nitrite toxicity on anammox bacteria.
Nitrite
Reduction
Concetration
Operation Mode
in activity
-1
(mg NO2 -N L )
Reference
100
100%
SBR*
(Strous et al., 1998)
350
50%
batch
(Dapena-Mora et al.,
2007)
75
28%
batch
(Bettazzi et al., 2010)
430
750
37%
100%
batch,
sludge embedded in
gel carrier
(Kimura et al., 2010)
400
50%
batch
(Lotti et al., 2012)
185
50%
batch
Chapter 3
384
50%
batch
Chapter 4
Decrease in
Full scale nitritationnitrogen
anammox
removal
*SBR: sequencing batch reactor
Overload
(van der Star et al., 2007)
The available reports on NO2- inhibition of anammox bacteria are based on batch
experiments which utilize biomass from nursing reactors, or observations made on
continuous bioreactors, but little is known about the sensitivity of anammox bacteria to
NO2- after being subjected to starving conditions. Starvation can occur in anammox
bacteria in underloaded bioreactors or during storage of sludge. Due to the slow growth
137
of anammox bacteria, new bioreactors are usually started up with enriched biomass from
other wastewater treatment plants (Joss et al., 2009, Vlaeminck et al., 2012, Wett, 2006),
and the biomass is often stored, remaining inactive during weeks or months. Although
studies have been carried out to optimize storage conditions (Vlaeminck et al., 2007),
there is no reports on the effect of the starvation on the tolerance of the bacteria to NO2-.
In this work we evaluated the inhibitory effect of NO2- on an anammox
enrichment culture subjected to different degrees of starvation. The impact of NO2toxicity and starvation were evaluated by studying the specific anammox activity of fresh
and starved biomass after treatments of NO2- exposure, as well as the evolution of the
ATP content of the biomass during such exposure events. Furthermore, we explored the
resilience of continuous anammox bioreactors to events of NO2- exposure, operated under
different nitrogen loading rates (NLR).
138
6.3
6.3.1
Materials and Methods
Batch bioassays
Anammox granular sludge (Chapter 5) was used in all the experiments. Batch
activity tests were performed in duplicate and incubated in an orbital shaker (160 rpm) in
a dark climate controlled room at 30 ± 2 ºC. The serum flasks (160 mL) were supplied
with 100 mL basal mineral medium (Sun et al., 2011) and inoculated with 0.71 g VSS L-1
of anammox granules. The basal medium was buffered with NaHCO3 (4 g L-1).
Subsequently the serum flasks were sealed with rubber stoppers and aluminum crimp
seals. The liquid and headspace were flushed with a gaseous mixture of He/CO2 (80/20,
v/v), leading to a pH ranging 7.1-7.3.
In experiments with “fresh biomass” the inoculum was withdrawn from the
nursing reactor immediately before the preparation of the experiments. In experiments
performed with “starved” biomass, the bottles were incubated in absence of N sources for
a defined period of time (starvation period), in serum flasks prepared as described above.
After the starvation period, the biomass was decanted, washed and replenished with
buffered fresh mineral medium. Then the flasks were sealed and purged with He/CO2 as
previously described.
139
The substrates were added by injection of concentrated solutions of NaNO2 and
NH4HCO3. In pre-exposure experiments, the bottles were supplemented with either NO2or NH4+, and incubated for a “pre-exposure period” of 24 h (resting cells) prior to
addition of the missing substrate. In simultaneous exposure experiments (metabolically
active cells), both substrates were fed together to the concentration desired in each
experiment.
6.3.2 Continuous bioreactors
Three laboratory-scale upflow anaerobic sludge blanket (UASB) reactors (500
mL) were operated in parallel. Each reactor was inoculated with 1.43 g VSS L-1 of
anammox granular sludge and incubated in a dark climate controlled room at 30 ± 2 ºC.
The reactors were fed with a basal mineral medium (described above), and operated
always at a hydraulic retention time of 0.25 d. The feeding media contained 4 g L-1 of
NaHCO3, and was flushed with He/CO2 (80:20; v:v) to make it anaerobic and provide a
pH of 7.2-7.3. The reactors were operated for 136 days. NO2- and NH4+ were fed to the
reactors a molar ratio of 1.2 (NO2- :NH4+). The NLR of the reactors was varied in four
different stages, 0.95 g N L-1 d-1 (0-42 d), 0.20 g N L-1 d-1 (43-82 d), 0.10 g N L-1 d-1 (83119 d) and again 0.95 g N L-1 d-1 (120-136 d). Three times during the operation of the
reactors (days 29, 78 and 120), the feeding of either NH4+ and NO2-, or both was
140
interrupted for 48 h. During these interruption events, R1 was fed with medium
containing no N compounds; the feed of R2 contained just 129 mg NO2- -N L-1, but no
NH4+, and the R3 did not receive any NO2-, and the concentration of NH4+ in the feeding
was 107 mg N L-1.
The performance of the reactors was evaluated by monitoring N2 production, and
the concentration of NH4+, NO2- and NO3- in the influent and effluent of the reactors, as
described in (Chapter 5). Furthermore, batch activity tests were periodically carried out
with inoculum collected from each reactor. The activity tests were carried out in 25 mL
serum flasks, with 14 mL of liquid volume. The preparation of the activity tests was done
as previously described.
6.3.3
Analytical methods
Nitrate (NO3-) and NO2- were analyzed by suppressed conductivity ion
chromatography using a Dionex IC-3000 system (Dionex, USA) fitted with a Dionex
IonPac AS18 analytical column (4 × 250 mm) and an AG18 guard column (4 × 50 mm).
During each run, the eluent (15 mM KOH) was used for 20 min. The flowrate was 1 mL
min-1.NH4+ was determined using a Mettler Toledo SevenMulti ion selective meter with a
Mettler Toledo selective NH4+ electrode (Mettler Toledo, USA). N2 was analyzed using
141
an Agilent7890 gas chromatograph (Agilent Technologies, USA) fitted with a Carboxen
1010 Plot column (30 m × 0.32 mm) and a thermal conductivity detector. The
temperatures of the column, the injector port and the detector were 220, 110 and 100°C,
respectively. Helium was used as the carrier gas and the injection volume was 100 µL..
The VSS content was analyzed according to Standard Methods (APHA, 2005). The ATP
extraction and analysis was performed as in the Chapter 5.
6.3.4
Assessment of specific anammox activity and inhibition
The specific anammox activity (SAA) was measured based on the N2 production
rate and expressed as g N g VSS-1 d-1. The SAA was calculated from the maximum slope
of the time course of the N2 concentration in the headspace as follows: (SAA) = ∆N2 (g
VSS ∆t)-1. The anammox activity in each assay was normalized with respect to the
activity of a control not subjected to inhibitory conditions, normalized anammox activity
(nAA, %) = (SAAinhibited/SAAcontrol) x 100. The concentration of NO2- causing 50%
inhibition (IC50) was calculated by interpolation in the graphs plotting the nAA as a
function of the NO2- concentration.
142
6.4
6.4.1
Results and Discussion
Effect of starvation on resistance of anammox resting cells to NO2- exposure
The sensitivity of anammox bacteria to the inhibitory effect of NO2- has been
shown to depend on the physiological status of the cells (Lotti et al., 2012, Scaglione et
al., 2012, Chapter 4). Different conditions, such as the absence of NH4+ (Chapter 4), or
the application of low pH in the medium ( Chapter 5), which interfere with the
mechanisms of generation of the metabolic energy available to the bacteria, have shown
to impact the tolerance of the bacteria to NO2-. Therefore, any factor that can impact the
energy status of cells may affect the capacity of the anammox bacteria to tolerate NO2-.
A set of experiments was designed to investigate how starvation could affect the
activity of anammox bacteria and their ability to resist inhibition during exposure to NO2in the absence of NH4+. Figure 6.1 shows the time course of N2 production of fresh and
starved anammox biomass, with different modes of substrate feeding including NH4+ (38
mg N L-1) pre-exposure, NO2- (50 mg N L-1) pre-exposure, and simultaneous feeding of
both substrates. Starved cells were highly inhibited by NO2- pre-exposure. After addition
of NH4+ , the SAA of starved, NO2- pre-exposed cells was 0.025 ± 0.010 g N g VSS-1 d-1.
In contrast, NO2- pre-exposure did not cause inhibition on fresh cells, which had an SAA
143
of 0.210 ± 0.006 g N g VSS-1 d-1 following NH4+ addition. The effect of starvation on the
SAA to cells simultaneously fed or just pre-exposed to NH4+ was minor. Therefore, the
starvation just compromised the ability of the cells to tolerate NO2- in the absence of the
Production N2 (mg N Lliq-1)
energy yielding substrate, NH4+.
60
40
20
0
0
2
4
6
Time (h)
8
Figure 6.1. Time course of N2 production of anammox granules pre-exposed
for 24 h to 50 mg NO2- - N L-1 (triangles), 38 mg NH4+ -N L-1 (squares), or
simultaneously fed (circles) after a starvation period of 0 d (close symbols) or
26 d (open symbols).
144
In order to investigate how fast the bacteria loose their capacity to overcome NO2toxicity under pre-exposure conditions, an experiment was set up after imposing
starvation periods from 0-43 d, prior to the pre- exposure treatments. In Figure 6.2, the
SAA of simultaneously fed, NH4+-pre-exposed and NO2- -pre-exposed granules is plotted
against the length of the starvation period applied before the pre-exposure treatments.
Anammox cells starved for longer times showed the lowest resistance to NO2- inhibition.
Most of the ability of the biomass to overcome NO2- toxic effect, was lost during the first
10 d of starvation, with a 64% decrease in activity after NO2- pre-exposure. The SAA of
NO2- pre-exposed cells further decreased to 90% after a starvation period of 26 d. On the
other hand, the anammox cells that were simustaneosly fed or NH4+-pre-exposed did not
lose N conversion capacity after starvation periods of up to 26 d, and their SAA remained
fairly constant, in a range 0.18-0.23 g N g VSS-1 d-1. For the highest starvation period (43
d), the SAA of the biomass in simultaneously fed- and the NH4+-pre-exposed treatments,
decreased by more than 50% when compared to the same experiments performed with
fresh biomass. This means that the integrity of the cells was compromised after imposing
starving conditions for too long, and therefore part of the inhibition observed under NO2exposure was due to death of cells in the enrichment culture, rather than just NO2- effect
on activity.
145
SAA (g N g VSS-1 d-1)
0.25
0.20
0.15
0.10
0.05
0.00
0
5
10
26
43
Starvation period (d)
Figure 6.2. SAA biomass pre-exposed to NO2- (50 mg N L-1) ( ), NH4+ (38
mg N L-1) ( ) or simultaneously fed with NO2- and NH4+ ( ), after
different periods of starvation.
Similar results were obtained with Nitrosomonas europaea, an NH4+ oxidizing
bacteria. Under NH4+ limitation, N. europaea lost their NH4+ oxidizing activity, due to
NO2- toxicity (Stein, Arp, 1998). Moreover the sensitivity of ammonia oxidizers to NO2was shown to be intensified in conditions of NH4+ starvation (Gerards et al., 1998).
146
6.4.2
Metabolically active and resting anammox cells respond differently to NO2inhibition after starvation
As shown in chapters 4 and 5, anammox bacteria are inhibited by NO2- through different
mechanisms depending on whether NH4+ is present or absent during the exposure to NO2. Therefore, the tolerance of metabolically active cells and resting cells to NO2- inhibition
may be impacted differently by starvation. A set of experiments was carried out in which
fresh and starved anammox cells were exposed to a range of NO2- concentrations in the
presence of NH4+ (50-500 mg NO2- -N L-1) or in a pre-exposure treatment (0-100 mg
NO2- -N L-1) prior to addition of the NH4+. In all these cases, the NH4+ concentration
during the monitoring period was 38 mg N L-1. In treatments where the concentration of
NO2- during the exposure period was lower than 50 mg N L-1, additional NO2- was
supplemented at the time of NH4+ addition to reach 50 mg N L-1 .
147
SAA (g N g VSS-1 d-1)
0.4
A
0.3
0.2
0.1
0.0
SAA (g N g VSS-1 d-1)
0
200
400
600
Concentration NO2- (mg N L-1)
0.4
B
0.3
0.2
0.1
0.0
0
20
40
60
80 100
Concentration NO2- (mg N L-1)
Figure 6.3. Effect of starvation on the activity of anammox cells exposed to NO2- in
presence of NH4+ (A) or pre-exposed to NO2- (B). Closed symbols represent fresh
biomass. Open symbols represent biomass starved for 20 d (A) or 14 d (B).
148
As shown in Figure 6.3A, the ability of starved-resting cells to overcome NO2pre-exposure was very limited when cells were starved. The 50% inhibiting concentration
(IC50) of NO2- to starved-resting cells was 7 ± 0 mg NO2- -N L-1, which is seven times
lower than the IC50 obtained with fresh (non-starved)-resting granules (52 ± 1 mg NO2- N L-1). On the other hand, when the pre-exposure took place in the presence of NH4+ , the
IC50 of starving cells was only 23% lower than the IC50 of fresh granules, which was 384
± 0 mg NO2- -N L-1 (Fig. 6.3B). The incubation in starving conditions did not affect the
integrity of the cells, since the maximum SAA of the biomass was conserved after the
respective starvation periods. The dramatic reduction in the resistance of anammox cells
to NO2- exposure under resting conditions, caused by starvation, suggests that their
strategy to mitigate the inhibitory effect of NO2- (reduce NO2- concentration in sensitive
region of the cell) relies on an internal energy source that is depleted during the starvation
period. The reduced availability of energy in starved resting cells, would therefore limit
their response to NO2- toxic concentrations. In contrast, when starved cells are actively
metabolizing during NO2- exposure, the bacteria are able to tolerate higher NO2- levels,
given that the N removal capacity is not seriously affected by starvation.
149
6.4.3
Effect of the starvation on the intensity of the response to NO2- stress
In Chapter 5, anammox bacteria were shown to actively respond to NO2- stress by
increasing intracellular ATP levels. A batch experiment was designed to analyze the
effect of starvation on the ATP response to NO2- exposure (100 mg N L-1) of anammox
resting cells. Starved cells produced less ATP than fresh cells (Fig. 6.4). The maximum
ATP content of starved cells was 1.54 times higher than initial. The peak was detected 1
h after addition of the NO2-. Likewise, the ATP content of fresh cells increased after
addition of the NO2-. After 1 h of incubation in the presence of NO2-, the ATP content of
the fresh cells was very similar to that of starving cells, but it kept increasing untill
reaching a maximum of 2.14 times higher than the initial value after 4 h of NO2exposure. These results are supportive of the idea that anammox cells invest energy to
mitigate NO2- toxicity. As shown before, starved cells, which generated less ATP, are
more sensitive to NO2- inhibition than fresh cells. Therefore, the more energy the bacteria
are able to recruit for detoxification, the more tolerant they would be to the presence of
toxic NO2-.
The results indicate that starved biomass is more sensitive to NO2- inhibition than
fresh biomass. Starvation may happen in continuous reactors, as a consequence of periods
150
of severe reactor underloading. Therefore, NO2- shocks during such periods, may cause
ATP Content (µ
µg ATP mg VSS-1)
serious instability.
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
2
4
6
8
Time (h)
Figure 6. 4. Evolution of the ATP content of anammox cells during a treatment of
exposure to 100 mg NO2- -N L-1, after a starvation period of 0 d ( ) or 19 d ( ).
6.4.4
Effect of sustained underloading on anammox bioreactors
Three continuous lab-scale anammox UASB reactors were utilized to evaluate the effect
of severe underloading on their ability to recover from NO2- exposure events. The three
151
reactors were initially operated at a NLR of 0.95 g N L-1 d-1. On day 29, the three reactors
were subjected to an event of substrate interruption, which lasted 48 h. The influent
conditions during this period were as follows: R1 was fed with basal medium containing
no N sources, R2 was feed contained 129 mg NO2--N L-1 d-1 as the only N source (Fig.
5), and R3 feeding contained 108 mg NH4+ -N L-1 d-1 as N source. During these 2 days of
substrate interruption no N transformation was observed in any of the reactors and as a
consequence, NO2- or NH4+ accumulated in the effluent of R2 or R3 respectively.
Immediately after reestablishment of the original feeding medium, on day 31, N removal
was completely recovered in the three reactors.
On day 43, the NLR of the reactors was reduced to 0.20 g N L-1 d-1 by decreasing
the concentration of NO2- and NH4+ in the feed. Substrates were again discontinued on
day 78, and during this event, 129 mg NO2- -N L-1 and 108 mg NH4+ -N L-1 were fed to
R2 and R3, respectively. After reestablishment of substrate feeding (0.20 g N L-1 d-1) on
day 80, full NO2- removal was recovered in the three reactors, and only the NH4+ excess
remained in the respective effluents. The NLR of the three reactors was further reduced
to 0.10 g N L-1 d-1 on day 83. The reactors were operated under these conditions for 37
days. Subsequently, a third similar event of substrate interruption was applied on day
120. NO2- (116 mg N L-1) and NH4+ (99 mg N L-1) accumulated in the effluents of R2
and R3 respectively. After 2 days of substrate discontinuing, the original medium (0.95 g
N L-1 d-1) was reestablished in the feeding of the three reactors. The N removal capacity
152
of R2 was severely reduced due to the exposure to NO2- alone during the feed
interruption event (Fig. 6.5A). Even after NH4+ was restored in the feeding of R2, both
NH4+ and NO2- accumulated in the effluent, indicating the anammox reaction was
severely disrupted. Slow recovery of the N removal could be observed and it attained
42% of the NLR on day 136, when the reactors were stopped.
In contrast, after substrate interruption and subsequent reestablishment of the
complete medium, full treatment capacity was recovered in R1 and R3. These results
indicate that sustained and severe underloading of the reactors increase the risk of failure
due to NO2- shocks.
The effect of underloading the reactors was further evaluated by performing
periodical batch activity assays with inoculum sampled from the three reactors. The SAA
of the biomass of each reactor was measured before and after each event of substrate
interruption. As shown in Figure 6.5B, exposure to either NH4+ or NO2-, or 2 days of
substrate starvation, during operation at 0.95-0.20 g N L-1 d-1, caused subtle changes in
the SAA of the biomass of the reactors. The large standard deviation of the measurements
on days 30 and 84 does not allow for further conclusions. Reducing the NLR from 0.95 to
0.20 g N L-1 d-1, caused a small decrease in the SAA of the granules, but a further
reduction in the NLR to 0.10 g N L-1 d-1 was not translated in an additional decrease of
153
the SAA. The failure of R2 after the event of the NH4+ interruption on day 120 could be
confirmed by the activity tests, which showed that the biomass was severely inhibited by
NO2-. In contrast, the granules in the R1 and R3 were not inhibited by the respective
events of substrate interruption (Fig. D1; APPENDIX D), but they benefited from the
increase in the NLR on day 120, after which the SAA increased 1.7- and 1.5-fold in R1
and R3, respectively.
The results obtained in the continuous reactors are consistent with the findings
from the batch experiments presented before. As shown in Figure 6.5, the sensitivity of
the biomass in R2 increased when the reactor was operated at low loading rates, which
are conditions expected to cause starvation. The lowest loading rate in R2 ranged 0.070.10 kg N kg VSS-1 d-1. Application of similar loading rates in an expanded granular
sludge bed reactor was reported to cause starvation of anammox bacteria, as evidenced by
a decrease in the heme group content of the biomass (Chen et al., 2013).
154
Concentration (mg N L-1)
140
A
120
100
80
60
40
20
0
0
SAA (g N g VSS-1 d-1)
0.8
20
40
60
80
100
120
140
20
40
60
80
100
120
140
B
0.6
0.4
0.2
0
0
Time (d)
Figure 6.5. Influence of NLR on the performance of anammox reactors subjected to
events of substrate interruption. A: Profiles of NH4+ and NO2- concentrations in the
influent and effluent of R2. NO2-inf (solid line), NH4+inff (dashed line), NO2-eff ( ) and
NH4+eff ( ). B: Evolution of the SAA of the biomass of the R1 ( ), R2 ( ) and R3
(▲).
155
6.4.5
Why does starvation affect the tolerance of anammox cells to NO2-?
The tolerance of anammox bacteria to NO2- is shown impacted by operational
conditions such as the absence of NH4+ (Lotti et al., 2012, Scaglione et al., 2012, Chapter
4) and the application of low pH values (Chapter 5). In absence of NH4+, the toxic effect
of NO2- is exacerbated. We have presented two hypotheses to explain the enhanced
sensitivity of anammox cells to NO2- when NH4+ is absent. Firstly, during active
metabolism the anammox reaction constitutes a continuous sink for NO2- , avoiding its
accumulation inside a sensitive region of the cells, where the inhibition happens (Chapter
4). In the absence of NH4+ the consumption of NO2- is not possible, and therefore NO2accumulates causing toxicity. Secondly, the anammox reaction is used to generate
metabolic energy in the form of an intracellular proton gradient between both sides of the
anammoxosome (van der Star et al., 2010). The transmembrane proton gradient
constitutes the driving force for generation of ATP enabling the proper functioning of
active transport proteins located in the anammoxosome membrane. NirC and NarK, are
two NO2- transporters which have been found in the genome of anammox bacteria
(Strous et al., 2006, van de Vossenberg et al., 2013). Although the function of these
proteins is not clear, they have been hypothesized to play an important role in the
resistance of anammox bacteria to NO2- inhibition, as they would actively pump toxic
NO2- out of the sensitive region of the cells to avoid inhibition (Chapter 4 and 5). The
primary energy source fueling active NO2- translocation is the anammox reaction, but in
156
absence of NH4+, the bacteria may recruit energy from an endogenous source. Anammox
bacteria have been reported to store glycogen in the riboplasm (van Niftrik et al., 2008).
The glycogen would be used as energy source for cell maintenance during starvation
periods, and its role providing energy to maintain the intracellular proton gradient, may
be of key importance for the resistance of anammox bacteria to NO2- toxic levels. As
shown by our results, resting anammox cells responded to NO2- stress by promoting the
generation of ATP (Fig. 6.4). Moreover, lower peak levels of ATP were observed in
starved cells, which were shown to suffer from serious inhibition.
NO2- causes inhibition to a wide variety of microorganisms, which have
developed different mechanisms of detoxification (Philips et al., 2002). Under conditions
of NO2- stress, some bacteria have been reported to invest energy to mitigate the
inhibitory effect. Denitrifying phosphorus accumulating organisms (DPAOs) and
Desulfovibrio spp. have been reported to utilize energy from intracellular energy storage
molecules to limit NO2- toxicity or mitigate cell damage (He et al., 2006, Zhou et al.,
2010). We suggest that when the anammox reaction does not occur, anammox bacteria
use the ATP to maintain the intracellular proton gradient, which is a driving force for
active NO2- transport out of sensitive regions of the cells . This mechanism of defense has
been previously described for bacteria suffering inhibition from weak protonophores
(weak acids)(Brul, Coote, 1999).
157
6.5
Implications
The tolerance of anammox bacteria to NO2- is very much influenced by the history of the
biomass. This partially explains the high divergence found in literature regarding NO2toxic levels. Anammox cells adapted to treating high loads of N are more resistant to
NO2- inhibition. On the other hand the ability of starved anammox biomass to withstand
NO2- shocks is seriously impaired. Therefore strategies must be followed to avoid severe
underloading of anammox bioreactors which may occur during dry weather conditions, if
bioreactors are oversized or in the treatment of low-strength wastewaters. If an anammox
biomass is to be stored due to its application to seasonal wastewaters, or to inoculate new
bioreactors, starvation of the inoculum during storage and transportation needs to be
minimized. Furthermore, during the start-up, accumulation of NO2- in the bioreactors
inoculated with starved biomass can cause the failure of the process. The anammox
bacteria are specially threatened by NO2- when exposure takes place in the absence of
NH4+. Therefore, control strategies need to be implemented to avoid complete oxidation
of NH4+ by nitrifiers, and guarantee a continuous source of NH4+.
158
CHAPTER 7 CONCLUSIONS
The anaerobic oxidation of ammonium is a novel and cost-effective
biotechnology for the treatment of ammonium rich wastewaters. One of the difficulties
inherent to the application of the anammox process to wastewater treatment is that due to
the slow growth of anammox bacteria, potential toxic events causing death of biomass
will require long recovery periods to reestablish full treatment capacity. Compounds
commonly found in wastewaters may pose a threat on the stability of the anammox
process. Among the substrates of the anammox reaction, nitrite, the necessary electron
acceptor of anammox, is of special concern and may completely inhibit the anammox
bacteria under certain conditions. The mechanism of anammox inhibition by nitrite has
not been described, nor the conditions under which anammox bacteria are more sensitive
to nitrite inhibition.
This research explores the inhibitory effect of several common wastewater
constituents on two different enrichment cultures of anammox bacteria. Both sources of
inoculum showed similar levels of inhibition by most toxicants studied. On the other
hand, PO43- stimulated the activity of the granular sludge, although it inhibited the
activity of the suspended enrichment culture at concentrations ranging 1.9 to 9.5 g L-1.
Sulfide, a product of biomass decay and sulfate reduction in anaerobic environments,
159
caused complete inhibition of anammox bacteria at concentrations as low as 10 mg H2S
L-1. Therefore, in effluents where sulfide is present, measures should be taken to remove
it prior to anammox treatment, e.g. by addition of iron (III) to precipitate sulfide. Oxygen
caused complete inhibition of anammox at 8 mg DO L-1. However, still considerable
anammox activity was observed at 2 mg DO L-1 which makes it feasible to accomplish
complete nitrogen removal in a single reactor combining anammox with partial
nitritation.
Among compounds involved in the anammox reaction, only NO3- and nitrite may
be found in wastewaters at concentrations potentially harmful to anammox bacteria. NO3caused moderate inhibition to both enrichment cultures. nitrite was highly inhibitory to
both inocula, with IC50 values of 151 and 185 mg N L-1, in the suspended and granular
cultures, respectively. The inhibitory levels of nitrite are of major concern in systems
were nitritation and anammox are carried out in separate units, where nitrite
concentrations fed to the anammox reactor are higher than in single reactor systems.
Special attention has been paid to nitrite. Although the inhibition of anammox
bacteria by nitrite has been widely reported, there is a great variability on the levels of
nitrite causing inhibition. One possible reason is that the tolerance of anammox cells to
nitrite, depends on the physiological status of the cells. We have developed a hypothesis
160
that anammox bacteria invest energy to avoid nitrite inhibition by actively transporting
this species away from a sensitive region of the cells. In this dissertation different
approaches have been used to disturb the availability of metabolic energy to the
anammox bacteria in order to assess its impact on the inhibition response to nitrite
exposure. Three approaches were followed. Firstly, the impact of nitrite on the activity of
metabolically active anammox cells (simultaneously fed with nitrite and ammonium) was
compared to the inhibitory effect caused during exposure to nitrite in the absence of
ammonium (resting cells). Secondly, the role of the pH on the resistance of metabolically
active- and resting anammox cells to nitrite inhibition was investigated. Thirdly, the
inhibitory effect of nitrite was evaluated on starved anammox cells. The findings
obtained from batch experiments were further demonstrated in continuous anammox
bioreactors.
The nitrite inhibitory effect was enhanced when the exposure took place in the
absence of ammonium. The IC50 value determined for nitrite was 7.2 times lower in
resting cells versus metabolically active cells. The inhibition in resting cells was found to
occur very quickly, with 74% loss of activity after only 30 min of exposure to nitrite. The
anammox activity was partially recoverable by washing the granules with nitrite free
medium, but most of the damage remained in the cells. Moreover, nitrite-containing
medium recovered after microbial incubation under ammonium deprivation was found to
161
cause toxicity to fresh healthy biomass, indicating that a toxic intermediate may be
generated during the exposure to nitrite.
The resistance of anammox cells to nitrite inhibition was impacted by the
application of low pH values. The inhibitory impact of nitrite on metabolically active
anammox cells was moderately enhanced when mildly acidic pH values were applied.
The effect of pH on the tolerance of resting cells to nitrite was much more pronounced,
e.g., complete inhibition was observed when the exposure to nitrite was conducted at pH
7.1, whereas no activity loss was observed cells exposure took place at pH 7.5. Although
previous studies have attributed nitrite inhibition to the undissociated free nitrous acid
(FNA), the results in this work demonstrate that FNA is poorly predictive of the
inhibition. Especially at the higher range of pH tested (7.3-7.8), the resistance of
anammox bacteria to nitrite inhibition was a function of the medium pH, irrespective of
the FNA concentration.
The inhibitory effect of nitrite was further investigated in starved anammox cells.
Starvation reduced the ability of anammox cells to tolerate nitrite. The loss in nitrite
tolerance during starvation occurred relatively quickly. Biomass starved for 10 and 26 d
showed a 64% and 90% inhibition after being exposed to 50 mg NO2--N L-1 in the
absence of ammonium, respectively. In contrast no inhibition was observed in parallel
162
experiments carried out with fresh biomass. Metabolically active and resting anammox
cells responded differently to nitrite inhibition after the starvation. The IC50 of nitrite in
resting-starved cells was 7 times lower than in resting-nonstarved cells. On the other
hand, the IC50 of nitrite in metabolically active cells only decreased 23% due to
starvation.
Nitric oxide (NO) gas was observed to accumulate in the headspace of
experiments with resting cells subjected to nitrite exposure. The amount of NO recovered
increased with the concentration of nitrite in the medium and the length of the nitrite
exposure period, and was dependent on the pH of the medium, i.e., the concentration of
NO measured in the head space of experiments carried out at pH 7.1 was 70 times higher
than at pH 7.5. NO generated during the nitrite exposure period was consumed
synchronously with the recovery of the anammox activity after addition of ammonium.
Moreover, during nitrite exposure of metabolically active cells, five-fold more nitrite was
needed to cause the same impact on NO production than with resting cells. In general,
higher NO accumulation corresponded with higher inhibition rates and, therefore, the
accumulation of NO gas was interpreted as an evidence of biochemical disruption in the
anammox cells.
163
Anammox bacteria were found to respond actively to nitrite stress. Analysis of the
evolution of the ATP content of the biomass showed temporal increase of ATP content in
cells after nitrite was supplied in the absence of ammonium. The observation of ATP
concentration peaks (with respect to time) were associated with conditions previously
shown to cause stress to the bacteria (evidenced by inhibition of the anammox activity
and accumulation of NO gas), indicating an active response of the bacteria to overcome
nitrite stress. Interestingly, the ATP response measured in starved cells was much lower
than in fresh cells, suggesting that the energy source used for the generation of ATP was
partially depleted during the incubation under starvation conditions.
The stability of continuous anammox reactors as influenced by the pH and
starvation was investigated. A bioreactor operated at pH 7.1 tolerated an event of
ammonium feeding interruption causing nitrite accumulation. Under these conditions, the
biomass was able to withstand nitrite exposure, and the reactor recovered full treatment
capacity as ammonium was reestablished in the feeding. In contrast, a parallel reactor
operated at pH ranging 6.4-6.7, was strongly impacted by the nitrite that accumulated
after depriving the ammonium feeding. Complete inhibition followed the nitrite exposure,
and N removal could not be recovered after ammonium was fed again to the reactor, nor
after readjusting the pH to 7.1.
164
Underloaded anammox bioreactors were shown to be more prone to failure due to
nitrite inhibition. When a reactor was operated at a N loading rate of 0.10 g N L-1 d-1,
nitrite exposure due to the absence of ammonium feeding caused inhibition of the
biomass in the reactor, which was translated in complete loss of the N removal capacity.
Partial recovery of the treatment capacity occurred slowly during the weeks following the
toxic event. On the other hand, when the reactor was operated 0.20-0.95 g N L-1 d-1, a
similar event of nitrite exposure was tolerated by the biomass and no signs of instability
were observed.
The work in this dissertation revealed different conditions under which anammox
bacteria are particularly sensitive to nitrite inhibition, i.e. absence of ammonium, mildly
acidic pH, and starvation. The mechanisms by which these conditions enhanced nitrite
toxicity were discussed. Firstly, ammonium is the electron donor of the anammox
reaction, enabling for active consumption of nitrite in actively metabolizing cells,
reducing its intracellular concentration to non-toxic levels. Therefore, if ammonium is not
available, nitrite will accumulate in sensitive regions of the cells, causing toxicity.
Secondly, we hypothesized that anammox bacteria avoid nitrite inhibition by pumping it
away from sensitive regions of the cells. Secondary active nitrite transporters - NirC and
NarK - have been found in anammox bacteria, which could play an important role in
detoxification of nitrite. These transport proteins rely on a proton motive force to actively
translocate nitrite. The absence of active metabolism, only possible in the presence of
165
ammonium, fueling the generation of an intracellular proton gradient, or the application
of low pH in the bulk medium, could disrupt the proton gradient that enables active nitrite
transport and, therefore, limit the ability of the anammox bacteria to overcome nitrite
exposure. When the cells were exposed to nitrite in the absence of active metabolism, the
anammox bacteria recruited energy in form of ATP, which could be used to maintain the
intracellular proton gradient. When cells were starved, the ATP response was less intense
than in fresh cells, indicating that the endogenous energy source supporting the
production of ATP was partially depleted. Anammox bacteria have been found to
accumulate glycogen to be used for cell maintenance during periods of starvation.
Therefore, the depletion of the glycogen pool caused by starvation may compromise the
ability of starved cells to overcome nitrite inhibition.
The new insights about the mechanisms of nitrite inhibition, provided in this
work, enable the design of operation and control strategies to minimize the risk of failure
in anammox bioreactors. In systems where partial nitritation and anammox are physically
separated, ammonium feeding to the anammox reactor should be always ensured in order
to avoid exposure of the anammox biomass to nitrite in the absence of the electron donor.
Operation of the bioreactors at the lower range of pH tested (<7.2) is not recommended,
as anammox bacteria have shown more sensitive to nitrite than at mildly alkaline pH
values. Additionally, starved cells are less tolerant to nitrite than fresh cells. Therefore
when anammox bioreactors are started up with enriched inoculum from other plants, the
166
length of the storage and shipping period of the inoculum needs to be minimized.
Furthermore, the accumulation of nitrite inside anammox bioreactors during the start-up
should be avoided to minimize the chances of inhibition of the delicate starved anammox
inoculum.
167
APPENDIX A.
Supplementary Data for CHAPTER 3
Basal Medium
The basal mineral medium was prepared using ultrapure water (Milli-Q system;
Millipore) and contained the following compounds (mg l-1): NH4HCO3 (213.6), NaNO2
(246.4), NaH2PO4•H2O (57.5), CaCl2•2H2O (100), MgSO4•7H2O (200), NaHCO3
(2,500), and 1.0 mL l-1 of two trace element solutions. Trace element solution 1
contained (in mg l-1): FeSO4 (5,000), and ethylenediamine-tetraacetic acid (EDTA)
(5,000). Trace element solution 2 contained (in mg l-1): EDTA (15,000), ZnSO4•7H2O
(430), CoCl2•6H2O (240), MnCl2 (629), CuSO4•5H2O (250), Na2MoO4•2H2O (220),
NiCl2•6H20 (190), Na2SeO4•10H2O (210), H3BO3 (14), and NaWO4•2H2O (50).
Analytical methods
Nitrate (NO3-) and nitrite (NO2-) were analyzed by suppressed conductivity ion
chromatography using a Dionex IC-3000 system (Sunnyvale, CA, USA) fitted with a
168
Dionex IonPac AS18 analytical column (4×250 mm) and an AG18 guard column (4×50
mm). During each run, the eluent (10 mM KOH) was used for 20 min. NH4+ was
determined using a Mettler Toledo SevenMulti ion selective meter with a Mettler Toledo
selective NH4+ electrode (Mettler Toledo, Columbus, OH, UAS). N2 was analyzed using
a Hewlett Packard 5890 Series II gas chromatograph (Agilent Technologies, Palo Alto,
CA) fitted with a Carboxen 1010 Plot column (30 m x 0.32 mm) and a thermal
conductivity detector. The temperatures of the column, the injector port and the detector
were 220, 110 and 100°C, respectively. Helium was used as the carrier gas and the
injection volume was 100 µl.
Molecular characterization of the inocula
The anammox bacteria in the anammox suspended enrichment culture (SEC) and
the anammox granular enrichment culture (GEC) were characterized by generating a
clone library. Community genomic DNA was extracted using the FastDNA Spin Kit for
Soil (Qbiogene, Inc, Carlsbad, CA) (Sun et al., 2009). The presence of anammox bacteria
was confirmed by PCR using specifically designed PCR primer set to target the 16S
rRNA gene of anammox bacteria, PLA46F and AMX-820R, which was described in the
previous study (Sun et al., 2011). The purified PCR products with primers of PLA46F
169
and AMX-820R were cloned into plasmid vector pCR 2.1-TOPO using the TOPO TA
cloning system (Invitrogen, Carlsbad, CA) to build a clone library. The details of cloning
and sequence analysis have been described in the previous study (Sun et al., 2011). The
number of clones analyzed for each culture was determined using a rarefaction curve to
estimate the diversity as previously reported (Sun et al., 2011). The clones were clustered
into phylotypes on the basis of sequence similarity > 99%. Sequence data were aligned
with ClustalX, including 16S rRNA gene sequences from reference bacterial strains
(GenBank) and unique phylotypes recovered from anammox suspended enrichment and
granular sludge, and a tree was constructed using PAUP* version 4.0b10. Two unique
phylotypes were indentified, one for each enrichment culture, which had a very high
similarity (99.5% or higher) with the 16S rRNA gene sequence of species from the genus
Brocadia (Fig. A1). The sequences of these clones have been deposited in the GenBank
database. The GenBank accession numbers for the sequences used to prepare
phylogenetic trees are shown as follows: SEC - Candidatus Brocadia sp. enrichment
culture clone MBR-EC-1, accession # JQ691616; GEC - Candidatus Brocadia sp.
enrichment culture clone ANA-GR-4, accession # JQ691617; Candidatus Brocadia
caroliniensis strain, accession # JF487828; Candidatus Brocadia sp. 40, accession #
AM285341; Candidatus Brocadia sp. enrichment culture clone RAS-Ina-1, accession #
HM769652; Candidatus Brocadia sp. enrichment culture clone ODS-1, accession #
HM769653; Candidatus Brocadia fulgida, accession # DQ459989.
170
Figure A1.- Phylogenetic tree for the anammox bacteria identified detected in the
anammox enrichment cultures utilized in this study.
Calculation of the concentration of unionized H2S
The concentration of unionized H2S was calculated the equation below:
[H 2 S ] =
[Na2 S ]added
 VHS

K1
 H ·
+ −apH
+ 1
 VL 10

Where:
H is the dimensionless Henry’s constant for H2S at 30ºC, (0.4543),
VHS is the head space volume,
171
VL is the liquid volume,
Ka1 is the equilibrium constant of the first dissociation of H2S, (1.023·10-7).
References
Sun, W., Banihani, Q., Sierra-Alvarez, R., Field J.A., 2011. Stoichiometric and molecular
evidence for the enrichment of anaerobic ammonium oxidizing bacteria from
wastewater treatment plant sludges. Chemosphere. 84, 1262-1269.
Sun, W., Sierra-Alvarez, R., Fernandez, N., Sanz, J.L., Amils, R., Legatzki, A.,
Maier, R., Field, J.A., 2009. Molecular characterization and in situ quantification of
anoxic arsenite oxidizing denitrifying enrichment cultures. FEMS Microbiol. Ecol. 68,
72-85.
172
APPENDIX B.
Supplementary data for CHAPTER 4
Effect of pre-exposure to NH4+ in absence of NO2-
NO2- pre-exposure was studied and discussed in the manuscript. In order to
discard side effects, exposure to NH4+ was also studied. The biomass was incubated for
24h in presence of NH4+ (76 mg N L-1). After the pre-exposure period, NO2- was
supplemented (up to 100 mg N L-1). As shown in Figure B1, neither exposing the
anammox granular sludge to NH4+ , nor starving biomass for 24 h had a negative effect
on the SAA. Non-pre-incubated control had a SAA of 0.92±0.02 g N g VSS d-1 and NH4+
pre-exposed biomass showed an SAA of 0.90±0.00 g N g VSS d-1. The biomass starved
for 24 h, showed a SAA of 0.92±0.04 g N g VSS d-1.
173
Figure B1.- Time course of N2 production of non-pre-incubated biomass
( ), biomass pre-incubated in absence of N compounds ( ), and
biomass pre-exposed to NH4+ for 24 h ().
Denitrifying activity of the anammox granular sludge
The anammox granules were tested for denitrification. Batch experiments were
carried out were the biomass was incubated in presence of NO2- (100 mg N L-1) and
stoichiometric amounts of H2 gas or methanol, as electron donors for denitrification.
174
Production N2 (mg N L-1)
200
150
100
50
0
0
5
10
15
20
25
Time (h)
Figure B2. – Timec ourse of N2 production of non-pre-incubated biomass
( ), biomass incubated in presence of NO2- only ( ), biomass incubated in
presence of NO2- and H2 (∆) or methanol ().
As shown in Figure B2, the production of N2 by the anammox granular sludge
was not stimulated in presence of electron donors that could be potentially used by
denitrifiers. The N2 production by biomass incubated in presence of NO2- only, as well
the one incubated in presence of H2 or methanol, was almost inexistent. The NO
accumulation was also very similar in the three cases.
175
APPENDIX C.
Supplementary data for CHAPTER 5
Measurement of Bioluminescence
50 µL of ATP extract were added to 450 µL of reagent (ATP determination kit,
Life Technologies, USA) and immediately analyzed. Quartz cuvettes of 500 µL were
used. Bioluminescence was analyzed in a fluorescence spectrometer (Model LS-55,
Perkin Elmer, USA) equipped with a Total Emission mirror. The apparatus was set in
bioluminescence mode, (Total Emission Mirror = IN), with excitation source turned off.
Delay time = 0 s, Gate time = 180 ms, Cycle time = 200 ms, Flash Count = 1. The slits
for excitation (off) and emission were 15 and 20 nm, respectively; Response = 4s,
Interval = 1s. The photomultiplier was set to 900V for maximum sensitivity. (See Perkin
Elmer, Fluorescence Applications).
176
NO2- inhibition of active anammox cells
When NO2- and NH4+ were fed together, the inhibitory effect due to high NO2concentrations did not occur instantaneously, but the N2 production rate decreased
N2 Production (mg N Lliq-1)
progressively during the first 2.4 h (Fig. C1).
80
Increasing Inhibition
Steady N2 production
60
40
20
0
0
1
2
3
Time (h)
4
5
Figure C1.- Timecourse of N2 production of cells simultaneously fed with
NH4+(76 mg N L-1 ) and NO2- (400 mg N L-1 , circles; 600 mg N L-1 ,
squares; 800 mg N L-1 , triangles) at different pH values (7, close symbols;
7.4, patterned symbols; 7.8, open symbols).
177
APPENDIX D.
Supplementary Data for CHAPTER 6
The influence of NLR on the tolerance of anammox bacteria to NO2- exposure
was studied in anammox upflow bioreactors. Three reactors were operated in parallel
with decreasing NLR from 0.95 g N L-1 d-1 (days 0-43), 0.2 g N L-1 d-1 (days 43-83) and
0.1 g N L-1 d-1 (days 83-end). The reactors were subjected to one events of substrate
interruption during each of the NLR periods, on days 29, 78 and 121. During these
events, which lasted 48 h, R1 was fed with a medium containing no N sources, the
medium fed to R2 contained just NO2- (129 mg N L-1) as N source and R3 was fed with a
medium containing just NH4+ (108 mg N L-1 ). Reactors 1 and 3 were used as control
reactors (Fig. D1), to explore whether starvation during the substrate interruption events
would hinder the N removal capacity. R1 and R3 were never exposed to NO2- in the
absence of NH4+ . The removal capacity of the reactors 1 and 3 was not affect by
interruption of the substrates. The removal of NO2- was near 100% during the complete
operation period.
178
Figure D1.- Profiles of NH4+ and NO2- concentrations in the influent and
effluent of control reactors R1 and R3. NO2-inf (solid line), NH4+inff (dashed
line), NO2-eff ( ) and NH4+eff ( ).
179
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