1. 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

2. 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 NH 3, NO 2 -, NO 3 - in incubated water samples maintained at river temp. Quantification of ammonia and nitrite oxidizing bacteria concentrations.

3. 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 48 RRI Lt 38 Lt 34 Lt 28 Lt 24

4. Dissolved oxygen and net flow in the San Joaquin River, 2003

5. DO @ saturation

6. Dissolved oxygen and net flow in the San Joaquin River, 2004

7. 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, NH 3, NO 2 -, NO 3 - of water samples collected at mid-depth. Long-term BOD bottle tests –Monitor NH 3, NO 2 -, NO 3 -, 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

14. Nitrification Ammonia oxidizing bacteria (AOB) NH 3 + 1.5 O 2  NO 2 - + H 2 O + H + Nitrite oxidizing bacteria (NOB) NO 2 - + 0.5 O 2  NO 3 -

15. Nitrification Equations Mechaelis-Menton expressions were used for bacteria growth and nitrogen species transformations Separate expressions for AOB, NOB,NH 3, NO 2, NO 3 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

16. Growth of ammonia oxidization bacteria (AOB): Growth of nitrite oxidization bacteria (NOB): Concentration of total ammonia (NH 3 ): Concentration of nitrite (NO 2 - ): Concentration of nitrate (NO 3 - ): Model equations

17. Nitrification Kinetic Parameters

28. Influence of flow on NH 3 DO demand

29. Net flow and Stockton NH 3 Effluent Conc, 2004

30. 70 Percent of BOD is NBOD, 2003 data

32. 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.

33. OF, 2-11-04, NOB p1=8, p2=7, p3=3 p2 dil factor=64 MPN value=1.636 Cells/L=598,308 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

34. MPN Analysis

35. AOB population increase after 30 day BOD incubation

36. 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.

37. Example Real-time Data Curves 11 Mar BOD samples a few 21 Jan & 24 Mar samples Ct threshold

38. 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.

39. 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.

40. 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.

41. OF 4-19-04 RRI 2-11-04 OF 2-26-04 RRI 5-15-04

42. 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.

43. End

44. 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

45. 12 lb. wt. PVC Frame Fluorometer (SCUFA III) Multi-parameter sonde (YSI 600XL) Sonar Transducer Monitor GPS/Map Plotter Computer 12 V – 110 V Inverter Depth Sounder GPS Antenna 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 Peristaltic Pump Tubing inlet for sample collection