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)


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)


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

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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


• 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


• 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.


• 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


• 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.

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

the mobi aerator in the stockton deep water ship channel

Documents

san joaquin river gary

measurement of oxygen

river temp

incubated water samples

flow vs t7

net flow

vs t rri7

bacteria concentrations