The MOBI Aerator in the
Stockton Deep Water Ship Channel
Gary M. Litton
Russ Brown
Marshall Haueter
Stephanie Kong
Nitrification in the San Joaquin River
Gary M. Litton
Mark Brunell
University of the Pacific
Overview
• Investigation of low dissolved oxygen episodes observed during winter months.
• Measurement of oxygen demands and nitrogen species at 9 locations in the San Joaquin River near Stockton.
• Long-term measurements of NH3, NO2-, NO3
- in incubated water samples maintained at river temp.
• Quantification of ammonia and nitrite oxidizing bacteria concentrations.
Low Dissolved Oxygen Critical
Reach
San Joaquin River
Stockton Deep Water
Ship Channel of the San Joaquin River
Nitrification study sampling station
BS
FC
OF
Lt 48RRI
Lt 38Lt 34
Lt 28
Lt 24
Dissolved oxygen and net flow in the San Joaquin River, 2003
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10/21/02 11/10/02 11/30/02 12/20/02 1/9/03 1/29/03 2/18/03 3/10/03 3/30/03
Time (day)
Dis
so
lve
d o
xyg
en
co
nce
ntr
ati
on
(m
g/L
)
0
200
400
600
800
1000
1200
1400
1600
Flo
w (
cfs
)
DO (mg/L) 7-d ave. flow
The DO-Sag in the DWSC2/21/2003
0
1
2
3
4
5
6
7
8
9
10
-3 -1 1 3 5 7 9
Distance (mi)
Diss
olve
d O
xyge
n (m
g/L)
DWSC(40 ft deep)
San Joaquin Riv(15 ft deep)
Stockton WastewaterOutfall
DO @ saturation
0
2
4
6
8
10
12
14
1/1/2004 0:00 1/21/200 2/11/200 3/3/2004 0:00 3/24/200 4/15/200 5/9/2004 0:00 5/30/200 6/20/2004 7/11/2004
DO
at R
RI (
mg
/L)
-500
0
500
1000
1500
2000
2500
3000
Net D
aily
Flo
w a
bo
ve S
tockto
n O
utf
all
(cfs
)
DO
Flow
Dissolved oxygen and net flow in the San Joaquin River, 2004
Approach• Water samples collected at 9 stations during 2003-2004
during winter and spring• Parameter depth profiles of temp., DO, pH, EC• BOD, CBOD, chl a, ph a, NH3, NO2
-, NO3- of water
samples collected at mid-depth.• Long-term BOD bottle tests
– Monitor NH3, NO2-, NO3
-, DO – BOD, CBOD (assess viability)
• Measure ammonia and nitrite oxidizing bacteria (AOB, NOB) populations with MPN and Real-Time PCR techniques.
• Estimate kinetic parameters of nitrification using a two-step model
RRI 2-25-04
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
0 10 20 30 40
Time (d)
(BO
D, C
BO
D, N
BO
D m
g/L
)
BOD CBOD NBOD
2/11/04 BOD 20 day
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
BS FC OF LT 48 RRI LT 38 LT 34 LT 28
Station Location
BO
D (
mg
/L)
BOD CBOD NBOD
February 4, 2004
0
1
2
3
4
5
6
7
8
R1 R2 (outfall) R2a (OF) R3 R4 R5 (RRI) R6 R7 R8
Distance below outfall (mi)
Nit
rog
en C
on
c (m
g/L
)
Org N NH3 NO2 NO3
Nitrogen concentrations in the DWSC 3-10-04
0
0.5
1
1.5
2
2.5
3
3.5
-4 -2 0 2 4 6 8 10 12
Distance downstream of the City of Stockton wastewater outfall (mi)
Nit
rog
en
(m
g/L
as
N)
NH3 NO2 NO3
Nitrogen concentrations in the DWSC 7-14-04
0
0.5
1
1.5
2
2.5
3
-4 -2 0 2 4 6 8 10 12
Distance from the City of Stockton Wastewater Outfall (mi)
Nit
rog
en
(m
g/L
as
N)
NH3 NO2 NO3
0 2 4 6 8 10 12 14 16 180
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Time (d)
Nitr
ogen
Con
cent
ratio
n as
N (
mg/
L)
Nitrogen concentrations at OF and RRI (3-10-2004)
RRI
RRI
OF
OF
NH3
NO2
NO3
Nitrification
• Ammonia oxidizing bacteria (AOB)
NH3 + 1.5 O2 NO2- + H2O + H+
• Nitrite oxidizing bacteria (NOB)
NO2- + 0.5 O2 NO3
-
Nitrification Equations
• Mechaelis-Menton expressions were used for bacteria growth and nitrogen species transformations
• Separate expressions for AOB, NOB,NH3, NO2, NO3
• The same kinetic parameters were used for all data
• Kinetic parameters were temperature adjusted
• Model fit achieved with:– initial nitrogen species concentrations
– initial AOB and NOB concentrations
AOBAOBNHNH
AOBNHAOBAOB XkCK
XC
dt
dX
33
3
maxGrowth of ammonia oxidization bacteria (AOB):
NOBNOB
NONO
NOBNONOBNOB Xk
CK
XC
dt
dX
22
2
maxGrowth of nitrite oxidization bacteria (NOB):
)(33
33
max
NHNHAOB
AOBNHAOBNH
CKY
XC
dt
dC
Concentration of total ammonia (NH3):
)()(22
2
33
32
maxmax
NONONOB
NOBNONOB
NHNHAOB
AOBNHAOBNO
CKY
XC
CKY
XC
dt
dC Concentration of nitrite (NO2
-):
)(22
23
max
NONONOB
NOBNONOBNO
CKY
XC
dt
dC Concentration of nitrate (NO3
-):
Model equations
)(dconstant rategrowth AOB maximum : 1max AOB
)(dconstant rategrowth NOB maximum : 1max NOB
(mg/L)growth AOBfor constant saturation-half :3NH
K
(mg/L)growth NOBfor constant saturation-half :2NO
K
) of AOB/mg/L of (mg/L AOBfor yield specific : 3NHYAOB
) of NOB/mg/L of (mg/L NOBfor yield specific : 2NOYNOB
Nitrification Kinetic Parameters
0 2 4 6 8 10 12 14 16 18 200
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5NH3,NO2,NO3 concentrations OF (3-10-2004)
Nitr
ogen
Con
cent
ratio
n as
N (
mg/
L)
Time (d)
AOB = 300000 cells/L, NOB = 400000 cells/L
NH3
NO2
NO3
0 2 4 6 8 10 12 14 16 18 200
0.5
1
1.5
2
2.5
3
3.5
4
4.5NH3,NO2,NO3 concentrations RRI (3-10-2004)
Nitr
ogen
Con
cent
ratio
n as
N (
mg/
L)
Time (d)
AOB = 1.5e+006 cells/L, NOB = 900000 cells/L
NH3
NO2
NO3
0 5 10 15 20 25
0
1
2
3
4
5
6
7
8NH3,NO2,NO3 concentrations OF (2-10-2004)
Nitr
ogen
Con
cent
ratio
n as
N (
mg/
L)
Time (d)
AOB = 400000 cells/L, NOB = 800000 cells/L
NH3
NO2
NO3
0 5 10 15 20 25
0
1
2
3
4
5
6
7
8
9
10NH3,NO2,NO3 concentrations RRI (2-10-2004)
Nitr
ogen
Con
cent
ratio
n as
N (
mg/
L)
Time (d)
AOB = 500000 cells/L, NOB = 800000 cells/L
NH3
NO2
NO3
0 2 4 6 8 10 12 14 16 18 20
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5NH3,NO2,NO3 concentrations OF (6-14-2004)
Time (d)
Nitr
ogen
Con
cent
ratio
n as
N (
mg/
L)
AOB = 150000 cells/L, NOB = 2000 cells/L NH3
NO2
NO3
0 2 4 6 8 10 12 14 16 18 200
2
4
6
8
10
12x 10
6 AOB and NOB populations
Bac
teria
Con
cent
ratio
n (c
ells
/L)
Time (d)
AOB
NOB
0 5 10 15 20 250
1
2
3
4
5
6
Time (d)
NB
OD
(m
g/L)
NBOD comparisons 3-10-2004 at RRI
NBOD measured
NBOD from NO2,NO
3 production
NBOD from N model
0 5 10 15 20 250
1
2
3
4
5
6
7
8
Time (d)
NB
OD
(m
g/L)
NBOD comparisons 3-10-2004 at OF
NBOD measured
NBOD from NO2,NO
3 production
NBOD from N model
OF BOD 20 day
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
2/11/2004 2/25/2004 3/10/2004 3/24/2004 4/19/2004 5/14/2004 6/4/2004 6/14/2004
Date
BO
D (
mg
/L)
BOD
CBOD
NBOD
RRI BOD 20 day
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
2/11/2004 2/25/2004 3/10/2004 3/24/2004 4/19/2004 5/14/2004 6/4/2004 6/14/2004
Date
BO
D (
mg
/L)
BOD
CBOD
NBOD
Influence of flow on NH3 DO demand
0.0
10.0
20.0
30.0
40.0
50.0
60.0
10/21/02 12/10/02 1/29/03 3/20/03
Time (day)
DO
or
Th
eo
reti
ca
l N
H3-
DO
de
ma
nd
(mg
/L)
0
200
400
600
800
1000
1200
1400
1600
Flo
w (
cfs
)
NH3 in Stockton eff luent Theoretical NH3 DO demand in SJR 7-day ave. f low
SJR DO and Stockton NH3 Discharge
0
2
4
6
8
10
12
14
1/1/2004 0:00 1/21/200 2/11/200 3/3/2004 0:00 3/24/200 4/15/200 5/9/2004 0:00 5/30/200 6/20/2004 7/11/2004
DO a
t RRI
(m
g/L)
0
5
10
15
20
25
30
Stoc
kton
Effl
uent
NH3
(m
g/L)
DO
NH3
Net flow and Stockton NH3 Effluent Conc, 2004
y = 0.8432x - 1.8825R2 = 0.9835
0
5
10
15
20
25
0 5 10 15 20 25 30 35
BOD at 20 days (mg/L)
NB
OD
at
20 d
ays
(m
g/L
)
70 Percent of BOD is NBOD, 2003 data
0
5
10
15
20
25
Tim
e to
red
uce
NH
3 by
50%
(d)
Outfall
RRI
2003 2004
Nitrifying Bacteria Quantitation
• Most-Probable Number (MPN) analysis– Sampling covered 8 time periods from 2-11-04 to 6-14-
04, and 3 stations (OF, RRI, and midstream). Total of 50 samples.
– AOB: samples mixed with ammonia-containing culture media and diluted 12-fold, with 8 replicates.
– NOB: as above, but with nitrite-containing medium.– 9 week incubation period, followed by testing for nitrite
or nitrate.– Dilutions at extinction used to estimate cell numbers
using MPN table.
OF, 2-11-04, NOB
p1=8, p2=7, p3=3p2 dil factor=64MPN value=1.636
Cells/L=598,308
RRI, 2-11-04 30 day bod
p1=8, p2=7, p3=5p2 dil factor=1024MPN value=2.124
Cells/L=12,428,434
RRI
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
3,000,000
3,500,000
4,000,000
4,500,000
5,000,000
Cel
ls/L AOB
NOB
OF
0
100,000
200,000
300,000
400,000
500,000
600,000
700,000C
ells
/L AOB
NOB
MPN Analysis
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000
18000000
OF RRI
3-10-04
30 day BOD
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
45000000
50000000
OF RRI
6-4-04
30 day BOD
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
45000000
50000000
OF Lt 48 RRI
2-11-04
30 day BOD
AOB population increaseafter 30 day BOD incubation
Nitrifying Bacteria Quantitation
• Real-Time PCR:– Molecular biology method which detects the number of
AOB-specific gene copies in water sample.
– Pure cultures of nitrifiers used to produce standard curves for absolute quantitation.
– Cell number per liter estimates.
– Currently, standards are being developed. DNA has been extracted from all 50 samples. Data set not yet complete.
– BOD samples have been analyzed and show end values markedly higher than starting values, as with MPN.
Example Real-time Data Curves
11 Mar BOD samples
a few 21 Jan &24 Mar samples
Ct threshold
Nitrifying Bacteria Quantitation
• Real-Time PCR: work to be completed– Production of additional standard curves using
different species of nitrifiers.– Development of internal controls to assess PCR
efficiency.– Development of Real-Time assay for NOB.
Bacterial and Algal Community Analysis with T-RFLP
• Terminal Restriction Fragment Length Polymorphism (T-RFLP)– Molecular biology method which generates diversity
fingerprints from environmental samples.
– Permits estimates of bacterial species diversity over time and space. Also, allow for species identifications when done in combination with DNA sequencing.
– Potential for ‘tracking’ a body of water by searching for similar bacterial and algal fingerprints in different regions of river.
Bacterial and Algal Community Analysis with T-RFLP
• Current progress:– All 50 samples have been analyzed and
replicated for total bacterial community diversity and over 25 clones have been sequenced and identified.
– T-RFLP data not yet analyzed.
50 100 150 200 250 300 350 400 450 500 550 600
trf OF3 2-26-04trf…2.fsa 9 Blue 1.0000 8-27f/1492r MspI
2000
4000
6000
50 100 150 200 250 300 350 400 450 500 550 600
trf OF3 4-19-04trf…1.fsa 5 Blue 1.0000 8-27f/1492r MspI
2000
4000
6000
50 100 150 200 250 300 350 400 450 500 550 600
trf RRI2 2-11-04tr…1.fsa 2 Blue 1.0000 8-27f/1492r MspI
2000
4000
6000
50 100 150 200 250 300 350 400 450 500 550 600
trf RRI 5-15-04trf…1.fsa 11 Blue 1.0000 8-27f/1492r MspI
2000
4000
6000
OF 4-19-04
RRI 2-11-04
OF 2-26-04
RRI 5-15-04
Bacterial and Algal Community Analysis with T-RFLP
• Future:– Data analysis of T-RFLP and maximizing of bacterial
identification via cloning and sequencing.– Production of T-RFLP patterns for only nitrifiers.
Method has been developed by other workers.– Development of T-RFLP for algal species. Involves
development of primer sets targeting taxonomic groups of algae, such as greens, diatoms, dinoflagellates, etc.
• Potential for algal diversity estimates and identifications using a fast and simple procedure.
• Potential for determining sources of algal populations in DWSC and other areas.
End
Algae
Respiration (- DO)Photosynthesis (+DO)
Sediment Oxygen Demand (-DO)
Atmospheric reaeration (+ DO)
Bacteria
Utilization (- DO)
Ammonia
Carbonaceous Organic Matter
Ammonia, nitrate and other nutrients
Processes that influence DO in the San Joaquin River
12 lb. wt.
PVC Frame
Fluorometer(SCUFA III)
Multi-parametersonde
(YSI 600XL)
Sonar Transducer
Monitor
GPS/MapPlotter
Computer
12 V – 110 V Inverter
DepthSounder
GPS Antenna
Real-Time Measurements:
Coordinate locationWater depthInstrument depthWater temperatureElectrical conductanceDissolved oxygenpHChlorophyll aRhodamine WT dye TurbidityPosition tracked on navigation chart
Peristaltic Pump
Tubing inlet for sample collection