Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Location: San Pedro Ocean Time Series (SPOT) station (33°33′N, 118°24′W)

Samples for quantification of retinal oxime were collected at a six of depths within the euphotic zone (5-250m). Seawater was collected from each CTD depth using Niskin bottles and immediately filtered. Particulate samples were collected using in-line 0.2µm, 3µm and 10µm pore-size filters and a peristaltic pump (flow rate < 50 ml per minute), transferred into sterile cryovials and were immediately stored at -80 degrees C until analysis.

Pigments were extracted from the filters in 3 mL of methanol, BHT (butylated hydroxytoluene) was added and placed in a -20 degrees C freezer overnight. The retinal oxime was formed by the addition of hydroxylamine hydrochloride and irradiated under yellow light for 2 hours before analysis. Retinal oxime samples were analyzed by liquid chromatography/triple mass spectrometry (LC/MS/MS/MS). The LCMS system consists of a ThermoTSQ Quantum Access electro-spray ionization triple quadrupole mass spectrometer, coupled to a Thermo Accela High Speed Liquid Chromatography system.

For chlorophyll-a measurements, 100 microliters of the pigment extraction were diluted in acetone (50x dilution) and analyzed using a Turner 10AU fluorometer.

Bacterial production was estimated by incorporation of [3H] thymidine and [3H] leucine into DNA and protein, respectively, as earlier described (Simon & Azam, 1989, Fuhrman and Azam 1982).

Deployment: SPOT
Platform: RV Yellowfin and RV Nerissa
Platform Type:  vessel
Start Date: 09/14/2016
End Date: 07/11/2017

The SPOT sampling for the months of May and July were carried out on the research vessel Nerissa of the Orange County Sanitation District. This was because the RV Yellowfin was in the dry dock for maintenance and repairs. Unfortunately, the light intensity for those two months is missing due to problems with the sensor.

The LC-MS data was processed using LCQUAN quantitative software from Thermo Scientific.

Description from NSF award abstract:
Rhodopsins are the simplest energy-harvesting photoproteins and community metagenomics have revealed that their synthesis genes are ubiquitous throughout the world oceans. These include microbial rhodopsin (proteorhodopsin (PR)), which occur in an estimated 75% of marine bacteria and archaea in oceanic surface waters. The discovery of this abundant and widespread photoprotein in the surface ocean has challenged the notion that solar energy can only be converted into chemical energy for growth in marine ecosystems through chlorophyll-based photosynthesis. Although the potential of light-driven energy flux in ocean ecosystems through PR could be significant, the physiological and ecological functions of this type of rhodopsin remains undetermined, mainly due to the lack of a technique for a direct measurement of this photoprotein. To evaluate the ecological relevance of PR in the marine environment, the investigators have developed a new analytical technique to measure the concentrations of the light-sensitive pigment in the PR, the chromophore retinal. Because rhodopsins have a single retinal chromophore associated with the polypeptide opsin, the total number of retinal molecules is equivalent to the total number of PR.

This project will employ the PI's newly developed protocol to examine the effects of light, organic carbon and trace metals availability on PR and bacteriochlorophyll synthesis using field and laboratory manipulations. Such experiments will establish the impact of abiotic factors on the two known bacterial photoheterotrophic metabolisms. The laboratory studies will be complemented with the analyses of those pigments in field samples collected along spatial and temporal gradients in light intensity, organic carbon and trace metals in different oceanographic regimes. Gene expression patterns will be determined in concert with changes in retinal and bacteriochlorophyll concentrations and microbial growth responses in the field and in the laboratory. Therefore, the combination of observational and manipulative approaches, will address fundamental questions in regard to the impact of retinal-based photochemical energy transformation in the ocean, a process that still is not well understood.