This document summarizes research on biological nitrogen removal technologies for liquid livestock manure, including nitrification/denitrification and deammonification processes. It describes the operation of full-scale systems in North Carolina that achieved over 90% removal of ammonia and total nitrogen using nitrification/denitrification. It also discusses single-tank deammonification reactors that removed ammonia from swine wastewater with 100% efficiency while reducing aeration needs by 57% compared to traditional methods. The research isolated specialized bacteria that enabled these nitrogen removal processes to work efficiently even at low temperatures.

1. Treatment Technologies for Ammonia in Liquid Manure:
Nitrification/denitrification and Anammox Based
Deammonification
From Waste to Worth Conference
Denver, Colorado, April 4, 2013
Matias Vanotti, Patrick Hunt, Ariel Szogi - USDA-ARS, Florence, SC, USA
Jose Martinez - IRSTEA, Rennes Centre, France
Airton Kunz - EMBRAPA Swine and Poultry, Concordia, Brazil
Takao Fujii - Sojo University, Kumamoto, Japan
Kenji Furukawa - Kumamoto University, Japan

2. North Carolina produces
approximately 750 million
chickens, 40 million turkeys,
3.5 billion table eggs, and
19 million hogs per year.
Animal Manure – Surplus N and Ammonia Emissions
in many regions of USA (and the world)
Walker et al., Atmos. Environ. 38:1235-1246
Ammonia Emissions

3. Treatment Technologies for Nitrogen
Management in Liquid Manure
• Biological nitrogen removal (conversion of
ammonia into N2)
1. Nitrification-Denitrification

4. •2nd Generation system (lagoon replacement)
• Full-scale treatment of 5,200 finishing pigs in North Carolina
•Solid-liquid separation with polymer, then nitrification/denitrification to
remove the ammonia, and P recovery.
Bioresource Technology 100 (2009): 5406-5416

5. Solids
N
P
Biological nitrogen removal: Nitrification-
Denitrification
NITRIFICATIONDENITRIFICATION
NH4
+
NO 2
-
NO 3
-
N2
NO x
Organic -C
Organic -C
Organic -C
O2
O2

6. TREATED
EFFLUENT
PHOSPHORUS
SEPARATION UNIT
B
BLOWER
POLYMER
FLOCCULANT
P
DENITRIFICATION
CLARIFICATION
NITRIFICATION
RETURN
BIOLOGICAL SLUDGE
CONFINED
LIVESTOCK
SOLID-LIQUID
SEPARATION
UNIT
EXCESS
BIOLOGICAL SLUDGE
RECYCLE
PHOSPHATE
PRECIPITATE
SLUDGE
P
M
HOMOGENIZATION
TANK
LIME
DEWATERED SOLIDS,
BIOLOGICAL SLUDGE &
PHOSPHORUS SLUDGE
P
M
PREANOXIC BIOLOGICAL
NITROGEN REMOVAL UNIT
Modified Ludzack-Ettinger (MLE)
P
SEPARATED
LIQUID
TANK

7. Denitrification
Nitrification
Denitrification
uses soluble carbon
from manure

8. •3rd Generation system (2012)
• Full-scale treatment of 1,200-sow farrow-to-finish (producing 30,500
hogs/year) in North Carolina
•Solid-liquid separation using settling and polymer, then
nitrification/denitrification to remove the ammonia
Terra Blue Inc., Clinton, NC

9. 0
5
10
15
20
25
30
35
40
Feb
Ma
Apr
Ma
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Jan
Feb
WaterTemperature(o
C)
Daily MAX
Daily MIN
Average
To reduce cost, the 2nd generation system incorporated new
nitrifying bacteria sludge acclimated to low temperatures

10. 0 5 10 15 20 25 30
0
500
1000
1500
2000
0
10
20
30
40
50
HPNS; this invention
Marine; Furukawa et al.
(1993)
On-site domestic, Chiemchaisri
and Yamamoto (1993)
Municipal; Chudoba and
Pannier, (1994)
Enriched nitrifying for high-ammonia
digester; Shammas, (1986)
Livestock; Bae et al. (2001)
Municipal; Wild et al. (1971)
NitrificationRate
gN/m3
-reactor/d
Cold weather municipal; Andersson
and Rosen (1990)
y = 1.25 + 1.93x
r2
= 0.998
Temperature, o
C
mgN/gMLVSS/h
High Performance Nitrifying Sludge
(HPNS):
• Isolated from manure
• High activity at low temperatures
• Used for rapid start-up of plants
HPNS was deposited in Agriculture Research
Culture Collection (Peoria, IL ) : NRRL B-50289

11. To start-up the plant, the nitrification tank (230
m3) was seeded with 1 liter of HPNS. In 40
days, it reached the optimum removal rate of
100 kg ammonia-N/day

12. Operation Simplicity
Once a week, the operator measured the nitrifying biomass
and set the operation parameters for the week.

13. MLE Biological N Removal performance
Influent
Conc. (After Solids
Separation) (ppm)
Effluent
Conc. (ppm)
Efficiency
%
TKN 1,428 597 101 145 92.9
NH4-N 1,182 483 59 124 95.0
COD 8,906 4,933 1,016 529 88.6
NH4-N removal efficiency:
Warm Weather: 94.0%
Cold Weather: 96.0%

14. Ammonia emissions reduction with this system
compared to the anaerobic lagoon technology
Aneja et al., 2008. Atmospheric Environment, 42:3291-3300
Emissions reduction
Warm Season 94.7%
Cool Season 99.0%

15. TSS
N
P
Lagoon System
New System
The use of clean water to flush barns
improved the air quality in the barns, pig
health and economic returns
0
2
4
6
8
10
12
14
16
18
May
Jun
Jul
Aug
Sep
NH3Concentration
inBarnExhaustAir(ppm)
Lagoon System
New System
Avg Lagoon Sytem
Avg New System
NH3 in the barns

16. Benefits of cleaner environment on
animal production
** Data compares five pig production cycles obtained with lagoon technology
and five pig cycles obtained with the new technology (5200 pigs/cycle)

17. Treatment Technologies for Biological
Nitrogen Removal in Liquid Manure
1. Nitrification-Denitrification
2. Partial Nitritation-Anammox
(Deammonification)
Anammox

18. Deammonification ProcessTraditional Nitrogen Removal Processes
Uses external
carbon addition
for DN
（nitrification / denitrification）
NH4
＋
Anammox
NO2
－
N2↑
Biological N removal processes
nitrification
denitrification nitrification
denitrification
MeOH
Ｑ
4 Ｑ
（partial nitritation / anammox)
Uses endogenous
carbon for DN No carbon needed

19. Anammox: new shortcut for the
biological removal of nitrogen
NITRIFICATIONDENITRIFICATION
NH4
+
NO2
-
NO3
-
N2
NOx
Organic-C
ANAMMOX
Organic-C
Organic-C
O2
O2
Anammox bacteria for low-cost treatment of livestock
wastewater
•Isolated from swine manure
in North Carolina
Brocadia caroliniensis
Anammox = Anaerobic ammonium oxidation
• ~ Half the aeration cost than
traditional method

20. Anammox biomass was grown in a parent reactor in
Florence, South Carolina.
Brocadia caroliniensis was deposited in USDA Agriculture Research Culture
Collection (Peoria, IL): NRRL B-50286

21. Partial nitritation + Anaerobic Ammonia Oxidation (ANAMMOX)
Partial
Nitritation ANAMMOX
aeration
NH4
+ + NO2
-
50 %
NH4
+ in
-
N2
Treated
effluent
Closed
Anaerobic
Environment

22. • Two
sequencing
batch tractors
(SBR)
• Treatment of
swine
wastewater
(1400 mg N/L)
Anammox coupled with Partial Nitritation
Two-stage process:
Anammox SBR
Magrí, A.; Vanotti, M.B.; Szögi, A.A.; Cantrell, K.B. Partial nitritation of swine wastewater in view of its coupling with the
anammox process. Journal of Environmental Quality (2012 doi:10.2135/jeq2012.0092)
Partial nitritation SBR

23. 2010: anammox bacteria handled
under anaerobic conditions

24. Completely anaerobic
Chambers??

25. 2011: Single tank deammonification
process (nitritation-anammox)
Biofilm plastic carriers
Mixing of specialized bacterial
cultures to start the one-stage
process
Continuous flow,
aerated reactor

26. Single-tank deammonification process start-up:
1. Mix of nitrification sludge with anammox,
2. start aeration and wastewater flow
Nitrification
sludge HPNS
Anammox sludge
B. Caroliniensis
in the single-tank

27. Continuous flow, aerated fluidized
reactor with biofilm carriers 30% v/v
Single-tank deammonification reactor setup (5-L reactor)

28. Another Single-tank deammonification reactor setup ( 1-L)
Air
flowmeter
and valve
Return
Sludge
Single
tank
Clarifier
DO
monitor
pH
monitor

29. 0
100
200
300
400
500
600
700
1 6 11 16 21 26
NConcentration(mg/L)
days
NH4-Nin
NH4-Nout
NO2-Nin
NO2-Nout
NO3-Nin
NO3-Nout
I II III
I = synthetic effluent (~360 mg NH4-/L),
II = swine effluent ~340 mg NH4/L),
III = swine effluent ~600 mg NH4-N/L).
Nitrogen Removal in Single Reactor

30. Single tank deammonification
of swine wastewater
Stoichiometry obtained:
1 NH4 + 0.87 O2  0.45 N2 + 0.11 NO3 + 1.41 H2O + 1.18 H+

31. Deammonification of swine wastewater
Parameter SW
Ammonia removal rate
1034 mg N/L-
reactor/day
Ammonia removal efficiency 100%
Total N removal efficiency 89.1%

32. Single tank deammonification process
Theory (partial nitritation + anammox):
NH4
+ + 0.85 O2 → 0.44 N2 + 0.11 NO3
- + 1.41 H2O + 1.19 H+

33. Stoichiometric rates
NH4
+ + 0.87 O2 → 0.45 N2 + 0.11 NO3
- + 1.41 H2O + 1.18 H+
Theoretical
Results of this study using digested swine manure
Deammonification reaction reduced 57% of the
oxygen requirements and 100% of the carbon needs.
(Compared to nitrification-denitrification, from 2.0 to 0.85 mol O2 /
mol NH4)
NH4
+ + 0.85 O2 → 0.44 N2 + 0.11 NOx
- + 1.41 H2O + 1.19 H+
Reactor 1 (5-L)
Reactor 2 (1-L)

34. •
•
New Deammonification Process
for Manure Digester Effluents
Deammonification Treatment (compared to nitrification-denitrification)
•It is quick and efficient (high removal rate = 1 kg NH4-N/m3/day)
•Organic carbon is not needed (best option for anaerobic digestion effluents)
•Aeration needs reduced by 57% (lower operational costs)

35. Conclusions
• Nitrification-denitrification of swine wastewater
was optimized after solids-liquid separation using
a pre-anoxic design and a high performance
nitrification sludge (HPNS)
• Single-tank deammonification with anammox
was feasible. It may be a key technology for
efficient ammonia treatment in systems that
consume carbon for energy production (AD)

36. http://www.ars.usda.gov/saa/cpswprc

38. Odor reduction in the liquid (99.9%)
0
500
1000
1500
2000
2500
3000
ppb
Sample
Skatole
Homogenization Tank
Separated Water
Post Nitrogen Treatment
Post Phosphorus Treatment
Biological N treatment
Loughrin et al., JEQ 38:1739-1748

39. GHG Emissions reduced 97% with the
replacement of the anaerobic lagoon with
the aerobic system
4430
542
18
135
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
TonCO2-eq/year
Baseline (lagoon) EST Project Activity
CH4 N2O
Vanotti et al., Waste Management 28:759-766

40. Greenhouse Gas (GHG) emission
reduction using aerobic treatment
(nitrification/denitrification and composting)
Vanotti et al., Waste Management 28:759-766
Baseline (lagoon)
Project Activity

41. Dec
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Jan
Feb
0
300
600
900
1200
0
20
40
60
80
100
Nitrogen
Pig Weight
TotalPigWeight(lb*1000)
FlushNLoad(kg/d)
New systems need to be robust and withstand the large load
changes during production (data show three production cycles)

42. Single-tank operation
• Continuous flow
• HRT 0.4-0.8 days
• Return sludge 2Q
• Ambient temperature (22 oC)
• Aeration rate 60 mL/min (DO 0.5-
0.9 mg/L)
• Plastic media 40% v/v
• Reactor MLVSS ~2600 mg/L
• Influent ammonia 365 to 600 mg
N/L
• Carbonate alkalinity 1700 to 3000
mg/L
• Start-up with synthetic wastewater (10 days), then switch to swine
wastewater (Effluent of anaerobic digestion)

43. Brocadia sp. (Candidatus AM285341)
Uncultured planctomycete Pla 5GA-8 (GQ356125)
Uncultured bacterium Asahi BRW (AB456583)
Brocadia caroliniensisCandidatus
Brocadia fulgida (Candidatus EU478693)
Uncultured planctomycete HAuD-MB/2-35 (AB176696)
Brocadia anammoxidans (Candidatus AF375994)
Uncultured anoxic sludge bacterium KU1 (AB054006)
Anammoxoglobus propionicus (Candidatus DQ317601)
Jettenia asiatica (Candidatus DQ301513)
Uncultured planctomycete KSU-1 (AB057453)
Kuenenia stuttgartiensis (Candidatus AF375995)
Scalindua sorokinii (Candidatus AY257181)
100
100
100
8999
87
96
97
100
81
0.005
Similarity to Candidatus “B. caroliniensis” (JF487828)
Candidatus “Brocadia fulgida” 96%
Candidatus “Brocadia anammoxidans” 94%
Candidatus “Jettenia asiatica” 92%
Candidatus “Kuenia stuttgartiensis” 90%
Candidatus “Anammoxoglobus propionicus” 90%
Candidatus “Scalindua sorokinii” 86%

44. Single-tank reactor N removal
at various N concentrations and loads
0
10
20
30
40
50
60
70
80
90
100
0
200
400
600
800
1000
1200
1400
1600
1800
2000
0 100 200 300 400 500
Removalefficiency(%)
NH4loadorremovalrate(mgN/L-
reactor/d)
Influent NH4
+ concentration (mg N/L)
NH4 load (mg N /L/d) NH4 removal (mg N/L/d) % Efficiency