Advancements in
Denitrification Processes in
Aquaculture
Jaap van Rijn
Faculty of Agricultural, Food and Environmental
Quality Sciences
The Hebrew University of Jerusalem
P.O. Box 12
Rehovot 76100, Israel
Topics
-General introduction to denitrification
-Factors affecting denitrification
-Denitrification and phosphorus removal
-Denitrification and alkalinity
-Application of denitrification in aquaculture
* with external carbon sources
* with internal carbon sources
Topics
-General introduction to denitrification
-Factors affecting denitrification
-Denitrification and phosphorus removal
-Denitrification and alkalinity
-Application of denitrification in aquaculture
* with external carbon sources
* with internal carbon sources
Anammox
Nitrate reduction pathways
Assimilatory
(NO3
Organic N)
Nitrate
reduction
DNRA (dissimilatory nitrate
reduction to ammonia)
(NO3 NO2
NH4)
Dissimilatory
Heterotrophic
(organic C and e- donor)
Denitrification
(NO3-
NO2- NO
N2O
N2)
Autotrophic
(inorganic C and e- donor)
Respiratory processes as a function
of Redox potential
Carbon
source
O2
Sediment
H2O
Redox
potential
decrease
NO3
N2
SO4
H2 S
CO2
CH4
Topics
-General introduction to denitrification
-Factors affecting denitrification
-Denitrification and phosphorus removal
-Denitrification and alkalinity
-Application of denitrification in aquaculture
* with external carbon sources
* with internal carbon sources
Environmental factors controlling
denitrification
Oxygen
Organic carbon
Temperature
pH
Substrates
Wide diversity of species and therefore
tolerant to wide range of environmental factors
Factors underlying nitrite accumulation in
denitrifiers
* Oxygen concentrations
* Kinetics of reductases
* Carbon availability
* Type of carbon source
* Carbon starvation
* Light exposure
NO3-
NO2-
NO
N2O
N2
Factors underlying nitrite accumulation in
denitrifiers
* Oxygen concentration
* Kinetics of reductases
* Carbon availability
* Type of carbon source
* Carbon starvation
* Light exposure
Factors underlying nitrite accumulation in
denitrifiers
* Oxygen concentration
* Kinetics of reductases
* Carbon availability
* Type of carbon source
* Carbon starvation
* Light exposure
Topics
-General introduction to denitrification
-Factors affecting denitrification
-Denitrification and phosphorus removal
-Denitrification and alkalinity
-Application of denitrification in aquaculture
* with external carbon sources
* with internal carbon sources
EBPR scheme
(Enhanced Biological Phosphate Removal).
Aerobic / Anoxic
Anaerobic
[Ac] [PO4]
Settling
[CO2] [PO4]
Effluent
Influent
PHB
Poly-P
PHB
Poly-P
Glycogen
Glycogen
Return sludge
Poly-P
Poly-P
Waste sludge
Poly-P
van Loosdrecht et al. (1997)
Phosphate removal by a denitrifying
consortium derived from the FBR
Barak and van Rijn (2000a)
Phosphate accumulation in denitrifiers
Polyphosphate
Before exposure to phosphate
After exposure to phosphate
Topics
-General introduction to denitrification
-Factors affecting denitrification
-Denitrification and phosphorus removal
-Denitrification and alkalinity
-Application of denitrification in aquaculture
* with external carbon sources
* with internal carbon sources
Nitrification
NH3 +2O2 = NO3- + H+ + H2O
(Alkalinity loss = 1 meq of alkalinity per mmole NH4+)
Denitrification
2NO3- + [5H2] + 2H+ = N2 + 6H2O
(Alkalinity gain = 1 meq of alkalinity per mmole NO3)
Autotrophic denitrification (on H2S)
5H2S + 8NO3- → 5SO42- + 4N2 + 4H2O + 2H+
(Alkalinity loss = 2 meq per 5 mmole H2S)
5SO42- + [20H2] + 10H+
5H2S + 20H2O
(Alkalinity gain = 10 meq per 5 mmole SO42-)
Total alkalinity gain = 1 meq of alkalinity per mmole NO3-
Autotrophic denitrification (on HS-)
5HS- + 8NO3- + 3H+ → 5SO42- + 4N2 + 4H2O
(Alkalinity gain = 3 meq per 5 mmole HS-)
5SO42- + [20H2] + 5H+
5HS- + 20H2O
(Alkalinity gain = 5 meq of alkalinity per 5 mmole SO42-)
Total alkalinity gain = 1 meq of alkalinity per mmole NO3-
Topics
-General introduction to denitrification
-Factors affecting denitrification
-Denitrification and phosphorus removal
-Denitrification and alkalinity
-Application of denitrification in aquaculture
*with external carbon sources
*with internal carbon sources
Recirculating aquaculture system
(Wade et al., 1996)
Stripping
Fluidized Bed Column
Culture basin
Drum
Filter
Ozonation
Sludge +
Water
U-tube
Disadvantages of current recirculating
systems
* Nitrate accumulation
* Sludge production
* Phosphate accumulation
Topics
-General introduction to denitrification
-Factors affecting denitrification
-Denitrification and phosphorus removal
-Denitrification and alkalinity
-Application of denitrification in aquaculture
*with external carbon sources
*with internal carbon sources
Heterotrophic Denitrification
5CH3COO- + 8NO3- + 3H+ 10HCO3- + 4N2 +
4H2O
C/N ration 1.25
Denitrifying reactors operated with
external carbon source
Type of denitrifying
reactor
Cultured
organisms
Carbon/
electron donor
Reference
Packed bed reactor (sw)
salmon
methanol
Balderston and Sieburth (1976)
Activated sludge (fw)
tilapia/eel
glucose/methanol
Otte and Rosenthal (1979)
Activated sludge (fw)
trout
corn starch
Kaiser and Schmitz (1988)
Packed bed reactor (sw)
flounder
glucose
Honda et al. (1993)
Packed bed reactor (sw)
squid
methanol
Whitson et al (1993)
Packed bed reactor (fw)
?
methanol
Abeysinghe et al. (1996)
Packed bed reactor (sw)
?
ethanol
Sauthier et al. (1998)
Packed bed reactor (sw)
shrimp
ethanol/methanol
Menasvesta et al. (2001)
Packed bed reactor (sw)
eel
methanol
Suzuki et al. (2003)
* fw = freshwater; sw = seawater
Menasvesta et al. (2001)
Summary of current status of denitrification in
recirculating systems
•Mainly applied in experimental systems
•Inducement of heterotrophic denitrifiers by
means of external carbon sources
•Use of submerged, packed bed reactors
Topics
-General introduction to denitrification
-Factors affecting denitrification
-Denitrification and phosphorus removal
-Denitrification and alkalinity
-Application of denitrification in aquaculture
*with external carbon sources
*with internal carbon sources
Denitrifying reactors operated with
internal carbon source
Type of denitrifying
reactor
Cultured
organisms
Reference
Activated sludge (fw)
carp
Meske (1976)
Digestion basin/
Fluidized bed reactor (fw)
tilapia
van Rijn and Rivera (1990)
Activated sludge (fw)
eel
Knosche (1994)
Packed bed reactor (fw)
Digestion basin/
Fluidized bed ractor (sw)
?
seabream
* fw = freshwater; sw = seawater
Phillips and Love (1998)
Gelfand et al. (2003)
Anaerobic carbon metabolism with nitrate as
terminal electron acceptor
Complex polymers
Hydrolytic bacteria
Mono + Oligomers
Propionate + Butyrate
Hydrogen producing
acetogenic bacteria
H2, CO2
Denitrifiers
CO2, N2, Bacterial biomass
Acetate
Nitrate
Ginosar
(Freshwater)
Rehovot
(Fresh and Marine)
Eilat
( Marine)
Intensive Freshwater Fish Culture Unit - Ginosar
Pumping basin
Fluidized bed reactors
Trickling
filter
Mechanical
Filter
Sedimentation
Basin
Water + Sludge
Water
Fish
Basins
Liquid
Oxygen
PO4-P (mg/l)
NO3-N (mg/l)
NO2-N (mg/l)
NH4-N (mg/l)
6
3
0
6
3
0
200
100
0
100
50
0
100
200
300
T im e (d )
400
Growth performance of tilapia in Ginosar
60
400
40
200
20
0
0
0
100
200
300
Days of growth
400
Average weight (g)
Fish density (kg/m3)
600
Fish yield and water use
Days of growth-----------------------
331.0
Biomass produced (kg)-------------- 4,868.0
Production (kg/m3)------------------
81.1
Average daily freshwater
addition (m3)--------------------------
2.9 (4.1%)
Water usage for fish
production (liters/kg) ----------------
190.0
Pilot-plant Marine Recirculating System
Foam
fractionator
Trickling filter
Settler
Fluidized bed
reactor
Pump
Pump
Fish basin
Digestion basin
NH4-N (mg/l)
5
20 ppt
15 ppt
10 ppt
5 ppt
4
0 ppt
3
2
1
0
NO2-N (mg/l)
10
5
0
NO3-N (mg/l)
150
100
50
0
0
100
200
D ay
300
400
Phosphate concentrations in fish basin
over experimental period
PO43--P (mg/l)
20
10
0
0
100
200
300
400
D ay
(Barak and van Rijn, 2000a)
Fish basin
Trickling Filter out
Sedimentation basin out
FBR out
Alkalinity (mg CaCO3/l)
180
160
140
120
100
80
60
40
20
0
7:00
11:00
Time (h)
15:00
I- Schematic Diagram of the DB Experimental Setup
DB out (to the Fluidized-Bed Reactor)
Aerial view
out
flow direction
intermediate
cross section
Top
Middle
Overlying liquid
Underlying sludge
in
Bottom
DB in (from the fish pool)
Working volume: 400 l
Retention time: ~1.5 hrs.
Flow rate: 5 l/min
IA- Chemical Parameters
Nitrate
Nitrite
8000
120
7000
100
NO2 (µM)
5000
80
60
-
4000
-
NO3 (µM)
6000
3000
2000
40
20
1000
0
0
T M
B T M
in
B T M
interm.
B
out
T
M
in
B
T
M B
interm.
T
M B
out
Total Ammonia
18000
16000
14000
12000
10000
8000
6000
4000
2000
Geochemical Processes
NH3 (µM)
•Nitrate/Nitrite Reduction
•Ammonification
•Dissimilatory Reduction of Nitrate
to Ammonia (DNRA)
150
100
50
0
T
M
in
B
T
M B
interm.
T
M B
out
IA- Chemical Parameters
Sulfate
Phosphate
1200
16000
1000
14000
PO4 (µM)
800
10000
600
3-
8000
2-
SO4 (µM)
12000
6000
4000
400
200
2000
0
0
T
M
B T
in
M B
T
interm.
M B
out
T
M
in
B
T M B
interm.
T
M B
out
Total Sulfide
7000
6000
H2S (µM)
5000
Geochemical Processes
4000
•Sulfate Reduction
•Sulfide Oxidation
•Organic Phosphorus Release
•Anaerobic Poly-P degradation
3000
2000
1000
20
10
0
T
M
in
B
T
M B
interm.
T
M B
out
IB- DGGE Analysis
Aqueous Samples
In Top
3.1
7.1
In Middle
3.1
7.1
Intermediate Intermediate
Top
Middle
3.1
7.1
3.1
7.1
Sludge Samples
Out Top
3.1
7.1
Out Middle
3.1
7.1
In Sludge
3.1
7.1
Intermediate
Sludge
3.1
7.1
Out Sludge
3.1
7.1
• DGGE of partial 16S rRNA gene fragments amplified with general bacterial primers –GCclamp-341F and 907R
• Samples run on an 18-55% DGGE gel gradient
1B- Phylogenetic Affiliations of Excized DGGE Bands
Excised Bands showing Closest “Blast” relative
Liquid
samples
Sludge samples
out
7.1.01 3.1.01
3.1.01
interme.
in
3.1.01
3.1.01 31.10.00
S2- Mesophilibacter aromativorans
(97%)
Bacteroidetes
S3- Flavobacteriales CF-1 (94%)
S4- Alkaliflexus imshenetskii (91%)
S5- Microscilla furvescens (90%)
S6A- Riftia pachyptila symbiont (93%)
S6I- Fusibacter paucivorans (94%)
S7- Desulfovibrio calendoniensis (98%)
S8- Marine Alpha Bacteria JP88 (97%)
S9- Bacterium from Denitrifying
Sludge Reactor AF234732 (97%)
S10- Dethioulfovibrio marinus (99%)
W4- Marine bacterium Keppib22 (96%)
W3- Marine alpha prot. AS-19 (99%)
W2- Microscilla furvescens (90%)
Fermentative
Bacteria
DB inlet (from fish basin)
Sulfate Reservoirs
from seawater
NH4
Nitrification
+
NO2-
CO2
Nitrification Products
from TF
NO3NO3-
Sulfide Oxidation
NO2-
NO
Rhodobacteraceae, Thauera
HSsulfur reduction
ammonification
Particulate Organic Carbon
Fish
excretions
N2
Oxygen-dependent
Thiomicrospira,
HS
symbiont-related SOB’s
Nitrate-dependent
Rhodobacteraceae
0
S
S0Dethiosulfovibrio, Fusibacter
Bacteroidetes
Fermentation Processes
CH2O
NH2COOCOO-R
Denitrification
SO4-
NH4
N2O CO2
NO
Sulfate reducers, 3
+ Clostridia, ?
-
DNRA CO2
Sulfate Reduction
HSDesulfovibrio, Desulfomicrobium
Desulfobacterium
SO4SO3-
CO2
Figure DD. Hypothetical model of of biogeochemical processes in the DB. Proposed bacterial
strains that participate in these processes are shown in italics. Dashed lines represent carbon
transformations.
(Cytryn, 2005)
Sulfur Transformations in the Fluidized Bed Reactor
Sulfide is extremely toxic to fish
• Blocks oxygen transport by hemoglobin
• Binds to the iron at the center of cytochrome molecules
Fluidized bed reactor
8/
20
/2
1
1
1
1
00
1
01
00
00
01
00
20
/2
/2
8/
27
16
8/
7/
7/
1
1
01
00
20
/2
/2
5/
26
15
7/
6/
6/
00
00
20
/2
/2
6/
24
11
6/
5/
5/
sulfide (µM)
/2
/0
/2
/0
/2
/2
/1
0/
01
00
00
00
00
99
99
01
20
20
20
20
20
19
19
/1
2/
9/
9/
2/
9/
3/
0/
12
03
11
05
04
02
12
11
sulfide (µM)
Sulfur Transformations in the Fluidized Bed Reactor
80
60
Rehovot 11/99-12/01
40
20
0
500
FBR Inlet
400
300
FBR Outlet
200
150
125
100
Eilat 5/01-8/01
75
50
25
0
(Cytryn et al., 2005)
DGGE Analysis of FBR community profiles
Dec.
` 99
Apr.
00
Nov.
00
Apr.
01
Dec. Wf Dec. Eilat
01
01
8/01
Phylogenetic Affiliation of Excized Bands- Proteobacteria
Sulfide-oxidizing
(autotrophic)
denitrifying bacteria
Filamentous
sulfide-oxidizing
bacteria
Hydrothermal vent eubacterium , U15103 Epsilon
Thiomicrospira denitrificans, L40808
proteobacteria
unidentified oil field bacteri, U46506
FBR3
Beggiatoa alba, L40994
Gamma
symbiont of Riftia pachyptila , M99451
proteobacteria
FBR5
marine humic oxidizing bacteri, AF521582
Thiothrix ramosa, U32940
Thiothrix sp. OS-F
FBR19
FBR20
Comamonas testosteroni, D87101 Beta
uncultured eubacteria ONG2
proteobacteria
FBR21
Hydrogenophaga taeniospiralis
uncultured sludge bacterium S1
FBR8
Thauera aromatica, X77118
Unknown Proteobacteria, X83533
FBR13
Alpha
Slope strain EI1
proteobacteria
Roseobacter denitrificans, M59063
FBR18
FBR17
uncultured Roseobacter Artic 9
FBE5
FBR14
FBR6
Paracoccus denitrificans , D13480
FBR7
Rhodovulum sulfidophilum, D16422
FBR15
Symbiotic sulfideoxidizing bacteria
Rhodobacteraceae
0.1
The fluidized-bed reactor (FBR)
N2
Sulfate
Autotrophic sulfide
oxidizers
5HS- + 8NO3- + 3H+ →
5SO42- +4N2 + 4H2O
Nitrate
Sulfide
Sulfur Transformations in the Anoxic Treatment Stage
Dissimilatory sulfate reduction
2
SO4
H2S
sulfate
sulfide
Chemotrophic
sulfide oxidation
S0
Elemental sulfur
Carbon, nitrogen and phosphorus in system
after 16 months growth period
Carbon Nitrogen Phosphorus
(as percentage of total feed input)
Retained in fish
18
15
21
Retained in system
11
12
79
Expelled from system
71
73
0
(Neori et al., in prep.)
Conclusions
• So far, denitrification has limited commercial application in
RAS systems
• Mainly external, not internal, carbon sources are used for
induction of denitrification in recirculating systems
• Additional benefits of denitrification:
*reduces/prevents organic matter discharge
*stabilizes buffering capacity
*prevents sulfide accumulation
*prevents phosphate accumulation
Acknowledgements
Faculty of Agriculture Rehovot
Dr. Yoram Barak
Dr. Eddie Cytryn
Dr. Yossi Tal
Mr. Iliya Gelfand
Mrs. Liat Koch
Mr. Gilad Fine
Mr. Yoni Sher
Volcani Institute
Dr. Dror Minz
National Center for Mariculture
Dr. Amir Neori
Leeds University
Prof. Mike Krom
University of Bayreuth
Prof. Andreas Schramm
Prof. Harold Drake
Mrs. Bärbel Krieger
MPI Bremen
Dr. Armin Gieseke
Dr. Dirk de Beer
Carsten Schwemer
Thank you!!!
Organic matter degradation
Performance parameters of prototype seawater,
recirculating system - Rehovot
________________________________________
Growth period
225.0
Initial average weight (g)
169.0
Final average weight (g)
378.0
Total fish biomass produced (kg)
58.1
Specific yield (kg/m3)
25.2
Survival
70.0
FCR
3.1
Average food input (g/day)
561.0
Freshwater supply (%)
1.3
Specific water consumption (l/kg)
109.0
________________________________________
“Atypical” Phosphate Removal by
Paracoccus denitrificans
PO4-P
Acetate
NO3-N
Glycogen
Total P
PHB
Barak and van Rijn (2000b)
Main physiological differences between Paracoccus denitrificans
and “polyphosphate accumulating organisms” (PAOs)
Organism(
s)
Anaerobic metabolism
PAOs
Use external carbon source forPHA synthesis
- Unable to use external carbon
P.
denitrifica source for PHA synthesis
ns
Aerobic/Anoxic metabolism
Grow and produce polyphosphate onPHA in absence of external carbon
source
- When present, external carbon source
might inhibit polyphosphate synthesis or
is used for PHA production but not for
growth
- Produces polyphosphate and grows on
energy provided by external carbon
source
- in absence of external carbon source,
cells with PHA do not grow and do not
produce polyphosphate