Subscriber access provided by OAKLAND UNIV
Article
The Confounding Effect of Nitrite on N2O Production by an Enriched Ammonia-Oxidising Culture Yingyu Law, Paul A. Lant, and Zhiguo Yuan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es4009689 • Publication Date (Web): 07 Jun 2013 Downloaded from http://pubs.acs.org on June 13, 2013
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Environmental Science & Technology
1
The Confounding Effect of Nitrite on N2O Production by an
2
Enriched Ammonia-Oxidising Culture
3
Yingyu Law1,2, Paul Lant3, Zhiguo Yuan1*
4 5
1
Advanced Water Management Centre, The University of Queensland, St Lucia 4072, Australia
6
2
Nanyang Technological University, SCELSE, Singapore 637551, Singapore
7
3
School of Chemical Engineering, The University of Queensland, St Lucia 4072, Australia
8
* Corresponding author: phone + 61 7 3365 4374; fax +61 7 3365 4726; email
9
[email protected] 10 11 12 13 14 15 16 17 18
ACS Paragon Plus Environment
Environmental Science & Technology
Table of Contents (TOC) art
19 20
22 23
N2O Biological NH2OH Oxidation NH3
NO2
NH 2OH Nitrifier Denitrification
-
NO N2O
24
N2O Production Pathways of
1.4 Step-wise Nitrite Dosing (DO 0.55 mg O2/L) Step-wise Nitrite Dosing (DO 1.30 mg O2/L) Step-wise Nitrite Dosing (DO 2.3 mg O2/L)
1.2 (mg N2O-N/ hr/ g VSS)
21
Specific N2O Production Rate
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 29
1.0
Nitrifier Denitrification
0.8 0.6
Biological NH2OH Oxidation
0.4 0.2
Ammonia-Oxidising Bacteria
25
0.0
N2O
N2O
N2O
0
200
26 NH4+ (~600-1400 mg N/L)
Nitritation
To Anammox Reactor
NO2- (~300700 mg N/L)
ACS Paragon Plus Environment
400 600 Nitrite (mg N/L)
800
1000
Page 3 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Environmental Science & Technology
27
Abstract
28
The effect of nitrite (NO2-) on the nitrous oxide (N2O) production rate of an enriched ammonia-
29
oxidising bacteria (AOB) culture was characterised over a concentration range of 0-1000 mg
30
N/L. The AOB culture was enriched in a nitritation system fed with synthetic anaerobic digester
31
liquor. The N2O production rate was highest at NO2- concentrations of less than 50 mg N/L. At
32
dissolved oxygen (DO) concentration of 0.55 mg O2/L, further increases in NO2- concentration
33
from 50 to 500 mg N/L resulted in a gradual decrease in N2O production rate, which maintained
34
at its lowest level of 0.20 mg N2O-N/hr/g VSS in the NO2- concentration range of 500-1000 mg
35
N/L. The observed NO2--induced decrease in N2O production was even more apparent at
36
increased DO concentration. At DO concentrations of 1.30 and 2.30 mg O2/L, the lowest N2O
37
production rate (0.25 mg N2O-N/hr/g VSS) was attained at a lower NO2- concentration of 200-
38
250 mg N/L. These observations suggest that N2O production by the culture is diminished by
39
both high NO2- and high DO concentrations. Collectively, the findings show that exceedingly
40
high NO2- concentrations in nitritation systems could lead to decreased N2O production. Further
41
studies are required to determine the extent to which the same response to NO2- is observed
42
across different AOB cultures.
43
Introduction
44
Ammonia-oxidising bacteria (AOB) are recognised as a major nitrous oxide (N2O) contributor
45
in wastewater treatment systems1-3. AOB generate energy through the aerobic oxidation of
46
ammonia (NH3) to nitrite (NO2-), typically described as a two-step process. NH3 is first
47
converted to NH2OH catalysed by a membrane bound ammonia mono-oxygenase (AMO),
48
followed by NH2OH oxidation to NO2- carried out by hydroxylamine oxidoreductase (HAO) in
ACS Paragon Plus Environment
Environmental Science & Technology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 29
49
the periplasm. N2O is produced by AOB as a by-product, possibly via one or more of the
50
following three pathways that have been proposed to date:
51
•
Nitrifier denitrification (also called autotrophic denitrification). This pathway involves
52
the sequential reduction of NO2- to nitric oxide (NO) and then to N2O by AOB, catalyzed
53
by the copper-containing NO2- reductase (NirK) and a haem–copper NO reductase (Nor),
54
respectively. NH3 4, NH2OH
55
potential electron donors. Nitrifier denitrification is active under aerobic conditions but is
56
particularly promoted under oxygen limiting and completely anoxic conditions 2, 9, 10.
57
•
5, 6
, hydrogen and pyruvate
7, 8
have been shown to be
Biological NH2OH oxidation. During NH2OH oxidation to NO2-, the nitrosyl radical
58
(NOH) and/or NO could be formed as intermediates 11. NOH can break down chemically
59
to form N2O
60
concerted or individual reaction of Nor, cytochrome c’-beta, cytochrome c-554 and
61
homologue NO reductases such as sNor
62
oxidation mainly takes place under aerobic conditions.
63
•
12, 13
, while NO can be subsequently reduced to N2O potentially by a
14
. N2O production via biological NH2OH
Chemical oxidation of NH2OH. NH2OH can be oxidized chemically to N2O by either
64
oxygen (NH2OH + 0.5O2 → 0.5N2O + 1.5H2O) or NO2- (NH2OH + NO2- + H+ → N2O +
65
2H2O)
66
occurs in the presence of a high concentration of NO2- (above 14 mg N/L) 16.
67
The amount of N2O produced, particularly from the nitrifier denitrification pathway
5, 15
. N2O production through chemical oxidation of NH2OH with NO2- usually
17, 18
, is
68
thought to largely depend on the concentration of NO2-. The expression of nirK in Nitrosomonas
69
europaea is directly regulated by a NO2--sensitive NsrR repressor protein whereby nirK is
70
expressed aerobically in response to increased NO2- concentration 18. In addition, the expression
71
of norB, which encodes NO reductase, was also elevated in N. europaea batch cultures
ACS Paragon Plus Environment
Page 5 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Environmental Science & Technology
72
supplemented with NO2- 19. Although NO2- is shown to promote the transcription of denitrifying
73
genes in N. europaea, contrasting effects of NO2- on aerobic production of N2O have been
74
observed in different AOB cultures. In a pure culture study on Nitrosomonas europaea, the
75
presence of NO2- was observed to be obligatory for aerobic N2O production 20, 21. In the presence
76
of ammonium (NH4+), NO2- was also shown to be a mandatory substrate for aerobic N2O
77
production by activated sludge
78
Knowles
79
production. The addition of NO2- did not increase the N2O production by N. europaea
80
physiological role of NO2- on the N2O production pathways of AOB is not clearly understood at
81
present and may differ from its stimulatory role at the gene transcription level.
82
26
22-25
. However, in their studies on N. europaea, Hynes and
and Ritchie and Nicholas
5
found that NO2- was not required for aerobic N2O 26
. The
NO2- can accumulate to high concentrations in the range of 300-700 mg N/L in nitritation 27
. Such high NO2- concentrations have been
83
systems treating high strength wastewater
84
postulated to lead to relatively high N2O emission factors of 1.2-10.5% of NH4+-N oxidised 28-31
85
for nitritation systems. However, a recent study by Law
86
emission factor of a lab-scale nitritation system was 0.5% of NH4+-N oxidised, despite the high
87
NO2- concentration in the system (550 ± 50 mg N/L). The emission factor is comparable to that
88
reported for domestic wastewater treatment plant and is approximately 6 times lower than
89
nitritation system in which nitrifier denitrification was proposed as the dominant N2O production
90
pathway 30.
91
32
reported that the aerobic N2O
The aim of this study is to elucidate the role of NO2- on N2O production by an AOB culture 32
92
enriched in a lab-scale nitritation system. The same nitrifying culture as used in Law et al.
93
used in this study. A wide NO2- concentration range of 0-1000 mg N/L is tested to provide a full
94
characterisation of the NO2- effects on N2O production by this culture. Since the N2O production
ACS Paragon Plus Environment
is
Environmental Science & Technology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 29
33
, the effect of NO2-
95
pathways of AOB can either be inhibited or promoted by oxygen
96
concentration is also examined at low to high dissolved oxygen (DO) levels.
97
Materials and Methods
98
Enriched AOB Culture. The experiments were carried out using an AOB culture enriched in an
99
8L lab scale sequencing batch reactor (SBR). The SBR was operated to perform nitritation in
100
identical cycles of 6 hours. The cycle consisted of 30 min settling, 10 min decanting, 2.5 min
101
feeding I (aeration on), 65 min aerobic reaction I, 92 min idle I, 2.5 min feeding II (aeration on),
102
65 min aerobic reaction II, 92 min idle II and 1 min wasting (aeration on). The mixed liquor
103
volatile suspended solid (MLVSS) concentration of the SBR was maintained at approximately
104
0.75 ± 0.05 g/L with a sludge retention time of 20 days. 2L of synthetic wastewater (composition
105
described below) was fed every cycle giving a hydraulic retention time (HRT) of 1 day. The
106
SBR was fed with synthetic wastewater mimicking characteristics of anaerobic digester liquor.
107
The daily nitrogen load was 1 kg N/m3/day. The composition of the wastewater (modified from
108
Kuai and Verstraete
109
KH2PO4 and K2HPO4 and 2 mL of a trace element stock solution. The trace element stock
110
solution contained: 1.25 g/L EDTA, 0.55 g/L ZnSO4·7H2O, 0.40 g/L CoCl2·6H2O, 1.275 g/L
111
MnCl2·4H2O, 0.40 g/L CuSO4·5H2O, 0.05 g/L Na2MoO4·2H2O, 1.375 g/L CaCl2· 2H2O, 1.25 g/L
112
FeCl3·6H2O and 44.4 g/L MgSO4·7H2O. The feed had a pH of 8.0 ± 0.1 and a molar ratio of
113
ammonium to bicarbonate of 1:1. 1 M NaHCO3 was used for pH adjustment at the end of each
114
aerobic phase to maintain pH above 6.4. The mixed liquor temperature was maintained at 33 ± 1
115
o
116
Law et al. 32.
34
) was: 5.63 g/L of NH4HCO3 (1 g/L NH4+-N), 0.064 g/L of each of
C using a water jacket. Details of the reactor design, operation and performance can be found in
ACS Paragon Plus Environment
Page 7 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Environmental Science & Technology
117
At the time these experiments were performed, the SBR had been operated for approximately
118
24 months and was displaying stable performance. Approximately 55% of the 1 g NH4+-N/L in
119
the feed was converted to NO2- at the end of a cycle. Minimal nitrite oxidising bacterial (NOB)
120
activity was detected with nitrate concentration lower than 20 mg NO3--N/L at all times.
121
Characterisation of the biomass composition using Fluorescence in situ Hybridization (FISH)
122
revealed that 81 ± 3% of the bacterial populations were ammonia-oxidising Betaproteobacteria,
123
which was dominated by Nitrosomonas sp. (67 ± 7%)
124
NSO1225, NSV443 35, NIT3 36; Ntspa662 37 and EUB-mix.
125
Batch Experiments. In each batch experiment, 1.1 L sludge was withdrawn from the SBR and
126
allowed to settle to a volume of 0.1 L. The settled sludge was then centrifuged at 4000 rpm for 3
127
mins and washed with NH4+ free synthetic medium (otherwise identical to the feed), pre-warmed
128
to 33 oC, the operational temperature of the SBR. The washed sludge was centrifuged and re-
129
inoculated into pre-warmed (33 oC) 1.0 L of NH4+ supplemented (550 mg N/L) synthetic
130
medium. Due to the high concentration of NO2- (550 mg N/L) in the mixed liquor, the
131
centrifugation steps were required to remove the NO2- present, to a concentration of < 5 mg N/L
132
with minimal biomass loss. To accommodate off-gas measurement, batch experiments were
133
carried out in a completely sealed 1.3L batch reactor with a gas outlet on the lid. Unless
134
otherwise stated, pH and DO were maintained at 7.0 ± 0.5 and 0.55 ± 0.05 mg O2/L,
135
respectively, conditions applied to the SBR. DO in the batch reactor was manually controlled
136
using a gas mixture of N2 and air. The N2 flow and air flow were adjusted using two mass flow
137
controllers (Smart- Trak 50 series- 1 L/min and 5 L/min, Sierra). In all batch experiments, the
138
total gas flow rate was maintained constantly at 1 L/min. pH was controlled automatically using
139
a programmable logic controller (PLC) system. During the course of the experiment, a mixture
32
. The probes used included NEU,
ACS Paragon Plus Environment
Environmental Science & Technology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
140
of ammonium bicarbonate (NH4HCO3) and NaHCO3 solution, with the NH4+ to HCO3- molar
141
ratio adjusted to 0.5:1 per L, was used to control the pH and to replenish NH4+. Since 2 moles of
142
HCO3- are required for the complete conversion of 1 mole of NH4+ to NO2-, using the pH control
143
system, NH4+ was able to be maintained relatively constant at 500 ± 30 mg N/L in all
144
experiments (Refer to Figure S1 in Supporting Information). With the exception of negative
145
control experiments whereby NH4+ was not supplied, 1 M NaHCO3 was used for pH adjustment.
146
Similar to the SBR, temperature was controlled at 33 ± 1 oC using a water jacket in all batch
147
tests.
148
The effect of NO2- was examined through the following 3 sets of experiments:
149
Set 1, individual NO2- dosing batch experiments. This set of experiments consists of six batch
150
tests with the addition of NO2- at 10, 20, 50, 100, 500 and 1000 mg N/L, respectively. Tests at all
151
levels were carried out in triplicates. At each NO2- dosing concentration, a negative control batch
152
experiment was also carried out whereby NH4+ was not supplied. In each test and negative
153
control experiment, NO2- was dosed at least 30 mins after the test started, when a pseudo-steady
154
state N2O production rate was observed. After NO2- dosing, the N2O production was monitored
155
for 30-40 mins.
156
Set 2, NO2- accumulation batch experiments. NO2- was allowed to accumulate gradually from 0
157
to 1000 mg N/L as a result of NH3 oxidation of AOB. The lack of NO2- oxidation activity
158
allowed NO2- to accumulate as long as NH3 oxidation activity was sustained. The N2O
159
production was monitored continuously for a period of 12-14 hours. The test was carried out in
160
duplicate.
ACS Paragon Plus Environment
Page 8 of 29
Page 9 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Environmental Science & Technology
161
Set 3, step-wise NO2- dosing batch experiments at varying DO concentration. NO2- was step-
162
wise dosed from 0 to 1000 mg N/L with a NO2- concentration increment of 10, 10, 30, 50, 400
163
and 500 mg N/L every 30 mins. This was conducted in three separate batch experiments with
164
DO concentration of 0.55, 1.3 and 2.3 mg O2/L, respectively. In a forth batch experiment, the
165
DO concentration was first step-wise increased from 0.55 to 1.3 and further to 2.3 mg O2/L every
166
30 mins. NO2- was then step-wise dosed with an increment of 10, 10, 30, 50, 400 and 500 mg
167
N/L every 30 mins.
168
Data Collection and Analysis. DO and pH in all experiments were continuously monitored
169
online using a miniCHEM-DO2 and a pH meter, respectively. The gas phase N2O concentration
170
was analysed with a URAS 26 infrared photometer (Advance Optima Continuous Gas Analyser
171
AO2020 series, ABB), with a dual measuring range of 0-100 ppmv and 0-1000 ppmv. The data
172
was logged every 3 sec. The analyser has a detection limit of 1% of the smallest measuring range
173
span. The 0-100 ppmv measuring range was used in all experiments giving rise to a detection
174
limit of 1.0 ppmv. A water trap was installed at the gas inlet of the analyser to prevent moisture
175
from entering the analyser. A t-shaped tubing joint was fitted onto the gas sampling tube
176
connecting the gas outlet of the reactor and the gas analyser. This allowed the excess gasflow
177
from aeration to escape from the system, maintaining atmospheric pressure in the headspace of
178
the reactor. The sampling pump of the analyser was adjusted to be lower than the total gas flow
179
rate in the reactor at all times.
180
Mixed liquor samples were taken periodically for NH4+, NO3- and NO2- analyses. Samples
181
were collected using a syringe and immediately filtered through disposable Milipore filters (0.22
182
µm pore size). The NH4+, NO3- and NO2- concentrations were analyzed using a Lachat
183
QuikChem8000 Flow Injection Analyzer (Lachat Instrument, Milwaukee). At the end of each
ACS Paragon Plus Environment
Environmental Science & Technology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
184
test, a mixed liquor sample was taken to determine the mixed liquor suspended solids (MLSS)
185
concentration and its volatile fraction (MLVSS). Each sample was analysed in triplicate
186
according to the standard methods 38.
Page 10 of 29
187
The N2O production rate was calculated by multiplying the measured gas phase N2O
188
concentration and the known gas flowrate. The average N2O production rate over each testing
189
period (with constant conditions applied) was calculated by averaging the measured N2O
190
production rate over the period (relatively constant in all cases). The NO2- production rate in
191
each testing period was determined through linear regression of the measured NO2- concentration
192
profile (mg NO2--N/L). The biomass-specific N2O production rate (mg N2O-N/hr/gVSS) and
193
biomass-specific NO2- production rate (mg NO2--N/hr/gVSS) were calculated by normalising the
194
N2O production rate and NO2- production rate data with the MLVSS concentration (g VSS/L).
195
The N2O emission factor was calculated based on the total amount of N2O emitted relative to the
196
total amount of NO2- produced (mg N2O--N/mg NO2--N) and is reported as a percentage.
197
Results
198
The Effect of Individual NO2- Dosing (Set 1 Experiments). An example batch test for each
199
NO2- dosing level of 10, 20, 50, 100, 500 and 1000 mg N/L is shown in Figure 1 a, b, c, d, e & f,
200
respectively. Prior to NO2- dosing, the specific N2O production rate in all the batch tests were
201
relatively high varying between 0.7-1.0 mg N2O-N/hr/g VSS at NO2- concentration of
