Lasers, Lights, and Phytoplankton: Laboratory Calibration of Optical Sensors from Autonomous Underwater Vehicles

Size: px

Start display at page:

Download "Lasers, Lights, and Phytoplankton: Laboratory Calibration of Optical Sensors from Autonomous Underwater Vehicles"

Gilbert Fisher
6 years ago
Views:

1 Lasers, Lights, and Phytoplankton: Laboratory Calibration of Optical Sensors from Autonomous Underwater Vehicles Diane Wyse, Moss Landing Marine Laboratories Mentor: James Bellingham Summer 2013 Keywords: LISST-100X, phytoplankton, multiple sensor data analysis ABSTRACT The purpose of this project is to compare the results from laboratory instruments and in situ sensors mounted on autonomous underwater vehicles when exposed to monocultures of phytoplankton. The project also focuses on calibration of in situ instruments using laboratory instruments and calibration beads to ground-truth the effort. Two species of phytoplankton within the size detection limits of the LISST-100X were cultivated in the Molecular Microbial Ecology (Worden) Lab at MBARI, and run on the suite of laboratory instruments. The results from this project describe the relationship between laboratory cell densities and ECO Puck chlorophyll concentration and cell densities for the LISST-100X ring intensity counts. INTRODUCTION Objectives: To ground truth the sensors mounted on the AUVs using monocultures of species of interest in the Monterey Bay via laboratory (ex-situ) culturing and testing. Typical Pseudo-nitzschia concentrations:

2 A four year sampling effort in MB showed max abundance of P. australis is highest between August and November (Buck et al, 1992). A study by Ryan et al (2005) using AUV surveys found maximum P. australis concentrations in northern MB around 2.5 x10^5 cells/l z = ~10 m, and around 1.5x10^5 cells/l (z = m) in central MB. Pseudo-nitzschia australis ranges from 75 t 144 um in length, with a valve width of 6.5 to 8 um (Buck et al, 1992). Questions: Can we compare data from the three sensors in this experiment? What sorts of relationships do we see, and can we identify organism signatures on the two AUV sensors to improve analysis of in-situ data? What signatures do we see for Micromonas sp., Ostreococcus, Heterosigma akashiwo? Five Species: Two that we can measure on the flow cytometer: Micromonas sp (< 2 um), Ostreococcus sp (2-3 um), Heterosigma akashiwo (18-34 um, oblong), Pseudo-nitzschia australis ( um), and Alexandrium catenella (l = um, w = um): (Fukuyo 1985; Fukuyo et al. 1990; Hallegraeff 1991; Taylor et al. 1995; Steidinger & Tangen 1996). MATERIALS AND METHODS CULTURE MEDIA AND MONOCULTURE INCUBATION Create culture media from seawater ~500 km offshore of Moss Landing (sampled along CalCOFI Line 67) The five monocultures grown over the course of this experiment were raised from Bigelow Lab strains provided by the Worden lab. The cultures were raised in F/2 growth media Establish light conditions in degree C incubator (~100 uein/m^2/s) INSTRUMENTATION Daily fluorescence readings were taken on a bench top Turner 10AU fluorometer between 0800 and 0930 for 54 days to establish growth curves and to determine the optimal chlorophyll concentration for each of the species. These measurements were taken to ensure that the phytoplankton were in their optimal growth phase, or on the upward slope of the curve, to test as

3 many healthy, viable cells as possible. After peak growth, cells begin to flocculate and die, and bacteria take over the culture. Accuri flow cytometer ECO Puck LISST-100X Isolate five plankton species to cultivate monocultures and graph their growth curves with time to determine optimal growth rate. Plots of Micromonas and Heterosigma growth curves for two generations before the and NEPCC.11 Strains were chosen to represent the range of smaller size classes of phytoplankton found in Monterey Bay. Two of these plankton species were tested on the instruments listed in the table below. Species Cell Width (µm) Strain Class Sensors Photo: T. Deerinck et al., and A.Z. Worden Micromonas sp. < 2 2.9RCC299 Prasinophyceae Turner 10-AU fluorometer, Accuri flow cytometer, ECO Puck, LISST-100X

Raphidophyceae Photo: WoRMS/Creative Commons Heterosigma akashiwo 18-34 NEPCC Turner 10-AU fluorometer, ECO Puck, LISST-100X, epifluorescence microscopy Optical Backscatter Forward Scattering

4 Raphidophyceae Photo: WoRMS/Creative Commons Heterosigma akashiwo NEPCC Turner 10-AU fluorometer, ECO Puck, LISST-100X, epifluorescence microscopy Optical Backscatter Forward Scattering Instrument 470 nm 650 nm Particle Scattering Distribution Chl- α Organism Count Cell Size Fluorometer Accuri flow cytometer Microscopy ECO Puck LISST-100X ECO Puck specifications: BB2FL-MBA-1073, measures optical scattering at 117 degrees at 470 and 650 nm wavelengths. The fluorometer (FL) gives a single-wavelength measurement of chl-

5 a, CDOM, phycocyanin, phycoerythrin, uranine (fluoescein), or rhodamine (source: WETLABS website and sensor description from shipment). Background on optics, experimental design, and tests performed to determine effectiveness of optically black material CHLOROPHYLL CONCENTRATIONS IN MONTEREY BAY A 2000 study by Wilkerson et al reports surface chlorophyll concentrations in the bay, sampled during spring (Mar, May) and fall (Sept, Nov) range from 0.07 to 6.09 ug/l for plankton size classes below 5 um, and from 6.92 to 95.2 ug/l for sizes above. This makes a case for testing larger organisms, and the contribution of the larger organisms to in situ chlorophyll concentrations. LISST 100X: 5 cm pathlength DATA ACQUISITION AND ANALYSIS LISST-100X data acquisition: The LISST-100X SOP software yields data in the form of both spherical and randomly shaped particle models. According to the manufacturer, when comparing output data to another laser particle sizer, the spherical particle model should be used, as other laser particle sizers use only spherical particle models. For this project, data was collected for both spherical and randomly shaped particle models for more complete data coverage and description of the nonspherical biological organisms. When the inversion calculation is applied, the data output will be converted to volume concentrations in ul/l for each size bin associated with the 32 detector rings. RESULTS

8 Instrument Micromonas sp. Heterosigma akashiwo ECO Puck (695 nm) 2E-8 µg Chl/L/cell 3E-6 µg Chl/L/cell

9 LISST Peak Channel 4E-6 Counts/cell Counts/cell DISCUSSION Data were recorded for optical backscatter measurements on the ECO Puck for Micromonas, Heterosigma, Ostreococcus, Isochrysis, and TruCount calibration beads. In all cases there is not a clear signal/noise, so further investigations should be performed to determine if the background can be further reduced or whether higher concentrations of the plankton are necessary for a detectable signal. CONCLUSIONS This study establishes a numerical relationship between each the ECO puck, LISST-100 and the laboratory cell counting instruments for Micromonas sp. and Heterosigma akashiwo. This lays the ground work for further investigations into establishing organism singature IDs on these instruments. Continuing research on this project will include performing an inversion to determine particle size distributions from the ring intensity count data on the LISST-100X. This data will be compared with data from the laboratory cell counts and with the ECO Puck data to determine if an organism signature can be identified and detected within in situ datasets. ACKNOWLEDGEMENTS I would like to thank Jim Bellingham for guidance through work with optical instruments, and for continued support of my thesis research. Thank you to Sebastian Sudek for assistance with cultivation and enumeration of phytoplankton in the Worden Lab. Thank you to members and affiliates of the Long-Range Autonomous Underwater Vehicle Lab, Brian Kieft, Thomas Hoover, Denis Klimov, and John Ryan for your help with location and design of bench-top experimental materials and software. Hans Thomas for used of the LISST-100X, Nilo Alvarado

10 for assistance with epifluorescence microscopy and instruction on DAPI. Thank you to Jian Guo for guidance on the bench-top fluorometer and the Accuri flow cytometer. Thank you to the members of the Worden Lab for welcoming me into your workspace and for the insightful thoughts and conversations. Thank you to the Packard Foundation and the Monterey Bay Aquarium Research Institute administration, staff, and facilities for your continued support of the internship program. Finally, I d like to thank George Matsumoto and Linda Kuhnz for their continued support and dedication to the internship program. References: Buck, K.R., L. Uttal-Cooke, C.H. Pilskaln, D.L. Roelke, M.C. Villac, G.A. Fryxell, L. Cifuentes, and F.P. Chavez (1992). Autoecology of the diatom Pseudo-nitzschia australis, a domoic acid producer, from Monterey Bay, California. Marine Ecology Progress Series, 84: Ryan et al, Physical biological coupling in Monterey Bay, California: topographic influences on phytoplankton ecology Wilkerson, F.P., R.C. Dugdale, R.M. Kudela, and F.P. Chavez (2000). Biomass and productivity in Monterey Bay, California: Contribution of the large phytoplankton. Deep-Sea Research II, 47: 1,003-1,021.

Field Instruments for Real Time In-Situ Crude Oil Concentration Measurements

Field Instruments for Real Time In-Situ Crude Oil Concentration Measurements Michael Sterling, Chris Fuller, Jim Bonner, Cheryl Page, and Gerardo Arrambide Texas A&M University and Conrad Blucher Institute