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Nitrification in the San Joaquin River. The MOBI Aerator in the Stockton Deep Water Ship Channel. Gary M. Litton Russ Brown Marshall Haueter Stephanie Kong. Gary M. Litton Mark Brunell University of the Pacific. Overview.

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The mobi aerator in the stockton deep water ship channel

Nitrification in the San Joaquin River

The MOBI Aeratorin the Stockton Deep Water Ship Channel

Gary M. Litton

Russ Brown

Marshall Haueter

Stephanie Kong

Gary M. Litton

Mark Brunell

University of the Pacific


Overview
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

Stockton Deep Water Ship Channel of the San Joaquin River

San Joaquin River

Lt 28

Lt 24

Lt 34

Lt 38

RRI

Lt 48

OF

FC

BS

Nitrification study sampling station


Dissolved oxygen and net flow

in the San Joaquin River, 2003



Dissolved oxygen and net flow

in the San Joaquin River, 2004


Approach
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


Nitrification
Nitrification

  • Ammonia oxidizing bacteria (AOB)

    NH3 + 1.5 O2 NO2- + H2O + H+

  • Nitrite oxidizing bacteria (NOB)

    NO2- + 0.5 O2 NO3-


Nitrification equations
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


Model equations

Growth of ammonia oxidization bacteria (AOB):

Growth of nitrite oxidization bacteria (NOB):

Concentration of total ammonia (NH3):

Concentration of nitrite (NO2-):

Concentration of nitrate (NO3-):




Net flow and Stockton NH3 Effluent Conc, 2004



Nitrifying bacteria quantitation
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.


RRI, 2-11-04 30 day bod

p1=8, p2=7, p3=5

p2 dil factor=1024

MPN value=2.124

Cells/L=12,428,434

OF, 2-11-04, NOB

p1=8, p2=7, p3=3

p2 dil factor=64

MPN value=1.636

Cells/L=598,308



AOB population increase

after 30 day BOD incubation


Nitrifying bacteria quantitation1
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 quantitation2
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
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 rflp1
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.


OF 2-26-04

OF 4-19-04

RRI 2-11-04

RRI 5-15-04


Bacterial and algal community analysis with t rflp2
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.



Processes that influence DO in the San Joaquin River

Respiration (- DO)

Atmospheric reaeration (+ DO)

Photosynthesis (+DO)

Algae

Ammonia, nitrate and other nutrients

Utilization (- DO)

Ammonia

Carbonaceous Organic Matter

Bacteria

Sediment Oxygen Demand (-DO)


GPS Antenna

GPS/Map

Plotter

Monitor

Depth

Sounder

Computer

Peristaltic Pump

12 V – 110 V Inverter

Sonar Transducer

Real-Time Measurements:

Coordinate location

Water depth

Instrument depth

Water temperature

Electrical conductance

Dissolved oxygen

pH

Chlorophyll a

Rhodamine WT dye

Turbidity

Position tracked on navigation chart

Multi-parameter

sonde

(YSI 600XL)

Fluorometer

(SCUFA III)

Tubing inlet for sample collection

PVC Frame

12 lb. wt.


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