Accepted Manuscript Microbial ecology of denitrification in biological wastewater treatment Huijie Lu, Kartik Chandran, David Stensel PII:
S0043-1354(14)00488-6
DOI:
10.1016/j.watres.2014.06.042
Reference:
WR 10755
To appear in:
Water Research
Received Date: 21 December 2013 Revised Date:
26 June 2014
Accepted Date: 29 June 2014
Please cite this article as: Lu, H., Chandran, K., Stensel, D., Microbial ecology of denitrification in biological wastewater treatment, Water Research (2014), doi: 10.1016/j.watres.2014.06.042. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
1
TITLE PAGE
2
Title:
4
Authors: Huijie Lu1*, Kartik Chandran2, David Stensel3
5
Affiliation:
7 8 9 10 11 12
1．Department of Civil and Environmental Engineering, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA
2．Department of Earth and Environmental Engineering, Columbia University, New York, NY 10027, USA
M AN U
6
RI PT
Microbial ecology of denitrification in biological wastewater treatment
SC
3
3．Department of Civil and Environmental Engineering, University of Washington, Seattle, WA 98195, USA Correspondent footnote:
Huijie Lu, Department of Civil and Environmental Engineering, University of Illinois at
14
Urbana Champaign, 205 N Mathews, Urbana IL 61801. Phone: (217) 333 8038, fax:
15
(217) 333 9464, email: [email protected]
AC C
EP
TE D
13
1
ACCEPTED MANUSCRIPT
16
Abstract Globally, denitrification is commonly employed in biological nitrogen removal processes
18
to enhance water quality. However, substantial knowledge gaps remain concerning the overall
19
community structure, population dynamics and metabolism of different organic carbon sources.
20
This systematic review provides a summary of current findings pertaining to the microbial
21
ecology of denitrification in biological wastewater treatment processes. DNA fingerprinting-
22
based analysis has revealed a high level of microbial diversity in denitrification reactors and
23
highlighted the impacts of carbon sources in determining overall denitrifying community
24
composition. Stable isotope probing, fluorescence in situ hybridization, microarrays and meta-
25
omics further link community structure with function by identifying the functional populations
26
and their gene regulatory patterns at the transcriptional and translational levels. This review also
27
stresses the need to integrate microbial ecology information into conventional denitrification
28
design and operation at full-scale. Some emerging questions, from physiological mechanisms to
29
practical solutions, for example, eliminating nitrous oxide emissions and supplementing more
30
sustainable carbon sources than methanol, are also discussed. A combination of high-throughput
31
approaches is next in line for thorough assessment of wastewater denitrifying community
32
structure and function. Though denitrification is used as an example here, this synergy between
33
microbial ecology and process engineering is applicable to other biological wastewater treatment
34
processes.
35
Keywords
36
Wastewater denitrification, microbial ecology, community structure, community function,
37
molecular biology
AC C
EP
TE D
M AN U
SC
RI PT
17
2
ACCEPTED MANUSCRIPT
38
Contents
39
1
Introduction ........................................................................................................................... 5 1.1
Biological denitrification and denitrifying microorganisms ............................................ 5
41
1.2
Wastewater denitrification ............................................................................................... 6
42
1.3
Molecular techniques characterizing denitrifying communities ...................................... 8
43
2
RI PT
40
Microbial ecology of wastewater denitrifying communities ........................................... 10 2.1
Overall diversity and dominant species ......................................................................... 10
45
2.2
Factors controlling community structure ....................................................................... 12
SC
44
2.2.1
Carbon sources ........................................................................................................ 12
47
2.2.2
Wastewater influent ................................................................................................ 15
48
2.2.3
Biofilm growth ........................................................................................................ 16
49
2.2.4
Operating conditions ............................................................................................... 18
2.3
TE D
50
M AN U
46
Factors controlling community function and the underlying mechanisms .................... 19
2.3.1
Carbon sources ........................................................................................................ 19
52
2.3.2
pH and temperature ................................................................................................. 20
53
2.3.3
Dissolved oxygen .................................................................................................... 21
54
2.3.4
Nitrogen oxides ....................................................................................................... 22
AC C
EP
51
55
3
Integrating microbial ecology information into process design, monitoring and
56
operation ...................................................................................................................................... 22
57
3.1
Integrating microbial ecology information into denitrification models ......................... 22
58
3.2
Improving system efficiency and stability ..................................................................... 24
59
3.3
Applying alternative carbon sources .............................................................................. 26 3
ACCEPTED MANUSCRIPT
60
3.4
Nitrous oxide emission control ...................................................................................... 28
61
3.5
Denitrification coupled to other processes ..................................................................... 29
4
Conclusions .......................................................................................................................... 31
63
References .................................................................................................................................... 40
RI PT
62
64
AC C
EP
TE D
M AN U
SC
65
4
ACCEPTED MANUSCRIPT
66
1
67
1.1
Introduction Biological denitrification and denitrifying microorganisms Biological denitrification is the sequential reduction of nitrate or nitrite to dinitrogen gas,
69
via the gaseous intermediates nitric oxide and nitrous oxide (Knowles 1982). This respiratory,
70
energy-generating process is catalyzed by four types of nitrogen reductases in sequence: nitrate
71
reductase (Nar), nitrite reductase (Nir), nitric oxide reductase (Nor) and nitrous oxide reductase
72
(Nos) (Zumft 1997). Over the last century, extensive research has been conducted on
73
denitrification for the following reasons. First, denitrification constitutes a main branch of the
74
biogeochemical nitrogen cycle, which returns reactive nitrogen to the atmosphere and maintains
75
the balance of the global nitrogen budget (Mike 2008). Second, as one of the important processes
76
to achieve biological nutrient removal (BNR), denitrification has been widely applied in
77
engineered wastewater treatment systems for targeted water quality improvement (Grady et al.
78
1999). Third, biological denitrification can contribute to the global greenhouse effect through the
79
emission of nitrous oxide (N2O), approximately 300 more potent than carbon dioxide (IPCC
80
2000).
TE D
M AN U
SC
RI PT
68
Although denitrification potentials are widely found in bacteria, archaea and some
82
eukaryote (e.g., fungi), nitrate reduction in natural and engineered ecosystems is primarily
83
conducted by bacteria (Knowles 1982, Cabello et al. 2004, Knowles 1996, Shoun et al. 1992).
84
Nutritionally, most denitrifying bacteria are facultative anaerobes using ionic and gaseous
85
nitrogen oxides as electron accepters in the absence of oxygen. The electron donors can be
86
derived
87
(chemolithoautotrophs). To date, only a limited number of chemolithoautotrophs are known to be
88
capable of denitrification (Sievert et al. 2008), whereas chemoorganoheterotrophic denitrifiers
AC C
EP
81
from
either
organic
(chemoorganoheterotrophs)
5
or
inorganic
compounds
ACCEPTED MANUSCRIPT
are distributed in a large variety of physiological and taxonomic groups (Knowles 1982). The
90
high phylogenetic diversity of heterotrophic denitrifiers is in parallel with their ubiquitous
91
presence in soil and aquatic habitats (Verbaendert et al. 2011, Shapleigh 2006). The
92
microbiology and ecology of denitrifying populations in these habitats has been extensively
93
investigated and reviewed (Knowles 1996). Due to their important roles in wastewater treatment
94
processes, denitrifying bacteria in engineered BNR systems are of particular interest to
95
wastewater engineers and microbiologists as a model microbial community.
96
1.2
SC
RI PT
89
Wastewater denitrification
With increased agricultural, domestic and industrial usage of nitrogen and phosphorus,
98
aquatic ecosystems around the world are facing severe water quality impairment caused by
99
nutrient enrichment (Smith et al. 1999). Notwithstanding the discovery and application of novel
100
chemolithoautotrophic denitrification processes such as anaerobic ammonia oxidation (Kuenen
101
2008), chemolithoautotrophic nitrification followed by chemoorganoheterotrophic denitrification
102
remains widely practiced for conventional biological nitrogen removal, where denitrification
103
occurs in the anoxic zone in the presence of no or limited dissolved oxygen (ideally
S0043-1354(14)00488-6
DOI:
10.1016/j.watres.2014.06.042
Reference:
WR 10755
To appear in:
Water Research
Received Date: 21 December 2013 Revised Date:
26 June 2014
Accepted Date: 29 June 2014
Please cite this article as: Lu, H., Chandran, K., Stensel, D., Microbial ecology of denitrification in biological wastewater treatment, Water Research (2014), doi: 10.1016/j.watres.2014.06.042. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
1
TITLE PAGE
2
Title:
4
Authors: Huijie Lu1*, Kartik Chandran2, David Stensel3
5
Affiliation:
7 8 9 10 11 12
1．Department of Civil and Environmental Engineering, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA
2．Department of Earth and Environmental Engineering, Columbia University, New York, NY 10027, USA
M AN U
6
RI PT
Microbial ecology of denitrification in biological wastewater treatment
SC
3
3．Department of Civil and Environmental Engineering, University of Washington, Seattle, WA 98195, USA Correspondent footnote:
Huijie Lu, Department of Civil and Environmental Engineering, University of Illinois at
14
Urbana Champaign, 205 N Mathews, Urbana IL 61801. Phone: (217) 333 8038, fax:
15
(217) 333 9464, email: [email protected]
AC C
EP
TE D
13
1
ACCEPTED MANUSCRIPT
16
Abstract Globally, denitrification is commonly employed in biological nitrogen removal processes
18
to enhance water quality. However, substantial knowledge gaps remain concerning the overall
19
community structure, population dynamics and metabolism of different organic carbon sources.
20
This systematic review provides a summary of current findings pertaining to the microbial
21
ecology of denitrification in biological wastewater treatment processes. DNA fingerprinting-
22
based analysis has revealed a high level of microbial diversity in denitrification reactors and
23
highlighted the impacts of carbon sources in determining overall denitrifying community
24
composition. Stable isotope probing, fluorescence in situ hybridization, microarrays and meta-
25
omics further link community structure with function by identifying the functional populations
26
and their gene regulatory patterns at the transcriptional and translational levels. This review also
27
stresses the need to integrate microbial ecology information into conventional denitrification
28
design and operation at full-scale. Some emerging questions, from physiological mechanisms to
29
practical solutions, for example, eliminating nitrous oxide emissions and supplementing more
30
sustainable carbon sources than methanol, are also discussed. A combination of high-throughput
31
approaches is next in line for thorough assessment of wastewater denitrifying community
32
structure and function. Though denitrification is used as an example here, this synergy between
33
microbial ecology and process engineering is applicable to other biological wastewater treatment
34
processes.
35
Keywords
36
Wastewater denitrification, microbial ecology, community structure, community function,
37
molecular biology
AC C
EP
TE D
M AN U
SC
RI PT
17
2
ACCEPTED MANUSCRIPT
38
Contents
39
1
Introduction ........................................................................................................................... 5 1.1
Biological denitrification and denitrifying microorganisms ............................................ 5
41
1.2
Wastewater denitrification ............................................................................................... 6
42
1.3
Molecular techniques characterizing denitrifying communities ...................................... 8
43
2
RI PT
40
Microbial ecology of wastewater denitrifying communities ........................................... 10 2.1
Overall diversity and dominant species ......................................................................... 10
45
2.2
Factors controlling community structure ....................................................................... 12
SC
44
2.2.1
Carbon sources ........................................................................................................ 12
47
2.2.2
Wastewater influent ................................................................................................ 15
48
2.2.3
Biofilm growth ........................................................................................................ 16
49
2.2.4
Operating conditions ............................................................................................... 18
2.3
TE D
50
M AN U
46
Factors controlling community function and the underlying mechanisms .................... 19
2.3.1
Carbon sources ........................................................................................................ 19
52
2.3.2
pH and temperature ................................................................................................. 20
53
2.3.3
Dissolved oxygen .................................................................................................... 21
54
2.3.4
Nitrogen oxides ....................................................................................................... 22
AC C
EP
51
55
3
Integrating microbial ecology information into process design, monitoring and
56
operation ...................................................................................................................................... 22
57
3.1
Integrating microbial ecology information into denitrification models ......................... 22
58
3.2
Improving system efficiency and stability ..................................................................... 24
59
3.3
Applying alternative carbon sources .............................................................................. 26 3
ACCEPTED MANUSCRIPT
60
3.4
Nitrous oxide emission control ...................................................................................... 28
61
3.5
Denitrification coupled to other processes ..................................................................... 29
4
Conclusions .......................................................................................................................... 31
63
References .................................................................................................................................... 40
RI PT
62
64
AC C
EP
TE D
M AN U
SC
65
4
ACCEPTED MANUSCRIPT
66
1
67
1.1
Introduction Biological denitrification and denitrifying microorganisms Biological denitrification is the sequential reduction of nitrate or nitrite to dinitrogen gas,
69
via the gaseous intermediates nitric oxide and nitrous oxide (Knowles 1982). This respiratory,
70
energy-generating process is catalyzed by four types of nitrogen reductases in sequence: nitrate
71
reductase (Nar), nitrite reductase (Nir), nitric oxide reductase (Nor) and nitrous oxide reductase
72
(Nos) (Zumft 1997). Over the last century, extensive research has been conducted on
73
denitrification for the following reasons. First, denitrification constitutes a main branch of the
74
biogeochemical nitrogen cycle, which returns reactive nitrogen to the atmosphere and maintains
75
the balance of the global nitrogen budget (Mike 2008). Second, as one of the important processes
76
to achieve biological nutrient removal (BNR), denitrification has been widely applied in
77
engineered wastewater treatment systems for targeted water quality improvement (Grady et al.
78
1999). Third, biological denitrification can contribute to the global greenhouse effect through the
79
emission of nitrous oxide (N2O), approximately 300 more potent than carbon dioxide (IPCC
80
2000).
TE D
M AN U
SC
RI PT
68
Although denitrification potentials are widely found in bacteria, archaea and some
82
eukaryote (e.g., fungi), nitrate reduction in natural and engineered ecosystems is primarily
83
conducted by bacteria (Knowles 1982, Cabello et al. 2004, Knowles 1996, Shoun et al. 1992).
84
Nutritionally, most denitrifying bacteria are facultative anaerobes using ionic and gaseous
85
nitrogen oxides as electron accepters in the absence of oxygen. The electron donors can be
86
derived
87
(chemolithoautotrophs). To date, only a limited number of chemolithoautotrophs are known to be
88
capable of denitrification (Sievert et al. 2008), whereas chemoorganoheterotrophic denitrifiers
AC C
EP
81
from
either
organic
(chemoorganoheterotrophs)
5
or
inorganic
compounds
ACCEPTED MANUSCRIPT
are distributed in a large variety of physiological and taxonomic groups (Knowles 1982). The
90
high phylogenetic diversity of heterotrophic denitrifiers is in parallel with their ubiquitous
91
presence in soil and aquatic habitats (Verbaendert et al. 2011, Shapleigh 2006). The
92
microbiology and ecology of denitrifying populations in these habitats has been extensively
93
investigated and reviewed (Knowles 1996). Due to their important roles in wastewater treatment
94
processes, denitrifying bacteria in engineered BNR systems are of particular interest to
95
wastewater engineers and microbiologists as a model microbial community.
96
1.2
SC
RI PT
89
Wastewater denitrification
With increased agricultural, domestic and industrial usage of nitrogen and phosphorus,
98
aquatic ecosystems around the world are facing severe water quality impairment caused by
99
nutrient enrichment (Smith et al. 1999). Notwithstanding the discovery and application of novel
100
chemolithoautotrophic denitrification processes such as anaerobic ammonia oxidation (Kuenen
101
2008), chemolithoautotrophic nitrification followed by chemoorganoheterotrophic denitrification
102
remains widely practiced for conventional biological nitrogen removal, where denitrification
103
occurs in the anoxic zone in the presence of no or limited dissolved oxygen (ideally
