Sara Thomas1 1UHAWAII
Objectives
The dark ocean (below 200 m) comprises ≥70 % of the global ocean’s volume, and contains ≥98 % of the global dissolved inorganic carbon (DIC; Gruber et al., 2004). While geochemical measurements have provided major advancements to understanding the general ocean carbon cycle, our knowledge of the dark ocean’s carbon cycle and microbial community is rudimentary (Herndl et al., 2005;
Reinthaler et al., 2010). Active and metabolically diverse microbial assemblages have recently been identified in the dark ocean (Herndl et al., 2005; Reinthaler et al., 2010; Swan et al., 2011) contradicting the historical view of dark ocean microbes as extremely slow growing or dormant. Investigating this active community may provide insights to the widely recognized uncoupling of primary production and respiration where microbial carbon demands exceed the particulate organic carbon flux below the euphotic zone. Over the past decade, major groups of Archaea, have been identified as a metabolically active fraction of the ocean’s mesopelagic and bathypelagic regions, and these nitrifying microorganisms appear to account for a significant fraction of inorganic carbon fixation in the ocean’s interior (Pearson et al., 2001; Herndl et al., 2005; Ingall et al., 2006). However, archeal nitrification appears insufficient to support measured inorganic carbon fixation rates (Agogué et al., 2008; Reinthaler et al., 2010; Varela et al., 2011) and so unidentified microbial lineages and energy sources are believed to contribute to a significant portion of dark carbon fixation (Swan et al., 2011). Genetic studies have identified novel genes contained in the dark microbial assemblage, including adenosine 5-phophosulfate reductase (aprA) (Meyer and Kuever, 2007; Swan et al., 2011), reverse-type dissimilatory sulfite reductase (rdsrA), and ribulose-1 5-phophosphate carboxylase-oxygenase (RuBisCO) (Swan et al., 2011). RuBisCO is found in two forms, with Form I the primary type observed in oceanic phytoplankton (Paul et al., 2000). However, in a study examining expression of the gene encoding the large subunit of RuBisCO,
Picard et al. (1997) unexpectedly found Form I rbcL genes actively expressed in the dimly lit regions of the euphotic zone and concluded chemoautotrophs, which utilize inorganic carbon, may also contribute to the diversity of carbon fixing organisms in the ocean. Furthermore, Swan et al. (2011) discovered consistent co-occurrence of aprA, rdsrA, and RuBisCO genes in multiple dark bacterial lineages, indicating the potential use of dissimilatory sulfur oxidation for energetic support of autotrophic carbon fixation in ubiquitous Deltaproteobacteria and Gammaproteobacteria (Swan et al., 2011). Given the abundance of these microbes in the dark ocean, this active community may fix inorganic carbon, coupled to the oxidation of reduced sulfur compounds, at globally significant rates providing new insights on carbon cycling in the global ocean.
The Polarstern ANT-XXIX/3 expedition provided the opportunity to explore the following questions:
1. How abundant are specific groups of chemoautotrophic bacteria in this region?
2. How do the relative distributions of these organisms vary along productivity gradients (onshore/offshore) and with changes in hydrographic forcing throughout the region?
Work at sea Sampling stations
Samples for marine microbiological investigations were collected from select stations during oceanographic/krill transects as seen in Figure 3.13.1.
Fig. 3.13.1: Stations sampled for marine microbiology
Nucleic acid collection and extraction
Seawater from discrete depths was collected from a Conductivity Temperature Depth (CTD) rosette into acid washed polycarbonate bottles. Two liters were pumped through 25 mm diameter 0.2µm pore size Supor ® filters and placed into 2 mL microcentrifuge tubes. Filters for RNA extraction were immersed in 500 µL RNlater buffer and flash frozen in liquid nitrogen when available on Polarstern.
All vials were stored at -80° C until processed in the shore-based laboratory in Honolulu, HI.
In the laboratory, plankton caught on filters will be mechanically and chemically disrupted to harvest nucleic acids. Briefly, DNA will be extracted from filters following a modified version of the Qiagen Plant kit. RNA will be extracted following a modified version of the Qiagen RNeasy procedure. DNA and RNA concentrations
will be quantified flourometrically using fluorometric dyes (Invitrogen, Carlsbad, CA, USA) and a plate reading spectrofluorometer.
Fluorescence in-situ hybridization (FISH)
Forty milliliters of seawater was also collected from discrete depths, placed into 50 mL centrifuge tubes, and fixed with formalin (2 % v/v final concentration). Fixed samples were held at 4° C for 24 hrs before being gently vacuum filtered (<5 mm Hg) onto a 25mm diameter 0.22 µm pore size polycarbonate filter supported by a glass-fiber filt er (GF/F). A small volume (5-10 mL) of Millipore water followed the seawater to wash the filters. Polycarbonate filters were attached to pre-labeled 75x25 mm microscope slides with tough-spot stickers or placed into shallow Gelman petri dishes with the appropriate GF/F. Processing in the home laboratory will follow the protocol described by DeLong et al. (1989) and Amann et al. (1990) to allow for quantification of specific groups of chemoautotrophic bacteria by fluorescence in situ hybridization (FISH).
Chlorophyll-a
To determine between station gradients in phytoplankton biomass, 125 mL of seawater from selected discrete depths was collected in an amber polycarbonate bottle and vacuum filtered onto a GF/F. Filters were folded in half, placed into a foil pocket and stored at -80° C. Upon processing in Honolulu, filters will be put into 5 mL acetone and allowed to extract for seven days in the dark. Chlorophyll concentrations will be quantified flourometrically using a Turner 10AU Fluorometer.
Data management
The relevant samples collected on expedition ANTXXIX/3 are intended for use within a Master’s degree research project at the University of Hawai’i at Manoa.
The study aims to publish in a peer-reviewed scientific journal to be determined at a later date.
Data will be provided upon request by the author.
References
Agogué H, Brink M, Dinasquet J, Herndl GJ (2008). Major gradients in putatively nitrifying and non-nitrifying Archaea in the deep North Atlantic. Nature, 456, 788-791.
Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. Journal of Bacteriology, 172(2), 762-770.
DeLong EF, Wickham GS, Pace NR (1989) Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science, 243(4896), 1360-1363.
Gruber N, Friedlingstein P, Field CB, Valentini R, Heimann M, Richey JE, Romero-Lankao P, Schulze D, Chenille C-TA(2004) The vulnerability of the carbon cycle in the 21st century;
An assessment of carbon-climate-human interactions. In: Field CB and Raupach MR (eds.) The Global Carbon Cycle: Integrating Humans, Climate, and the Natural World.
Island Press, Washington, D.C., pp 45-76.
Herndl GJ, Reinthaler T, Teira E, van Aken H, Veth C, Pernthaler A, Pernthaler J (2005) Contribution of Archaea to total prokaryotic production in the deep Atlantic Ocean.
Applied and Environmental Microbiology, 71(5), 2303-2309.
Ingall AE, Shah SR, Hansman RL, Aluwihare LI, Santos GM, Druffel ERM, Pearson A (2006) Quantifying archaeal community autotrophy in the mesopelagic ocean using natural radiocarbon. PNAS, 103(17), 6442-6447.
Meyer K, Kuever J (2007) Molecular analysis of the diversity of sulfate-reducing and sulfur oxidizing prokaryotes in the environment, using aprA as functional marker gene. Applied and Environmental Microbiology, 73(23), 7664.
Paul JH, Alfreider A, Kang JB, Stokes RA, Griffin D, Campbell L, Ornolfsdottir R (2000) Form IA rbcL transcripts associated with a low salinity/ high chlorophyll plume (‘Green River’) in the eastern Gulf of Mexico. Marine Ecology Progress Series, 198, 1-8.
Pearson A, McNichol AP, Benitez-Nelson BC, Hayes JM, Eglinton TI (2001) Origins of lipid biomarkers in Santa Monica Basin surface sediment: A case study using compound-specific D14C analysis. Geochimica et Comosochimica Acta, 65(18), 3123-3137.
Pichard SL, Campbell L, Paul JH (1997) Diversity of the ribulose bisphosphate carboxylase/
oxygenase form I gene (rbcL) in natural phytoplankton communities. Applied and Environmental Microbiology, 63, 3600-3606.
Reinthaler T, van Aken HM, Herndl GJ (2010) Major contribution of autotrophy to microbial carbon cycling in the deep North Atlantic’s interior. Deep-Sea Research II, 57, 1572-1580.
Swan BK, Martinez-Garcia M, Preston CM, Sczyrba A, Waoyke T, Lamy D, Reinthaler T, Poulton NJ, Masland EDP, Gomez ML, Sieracki ME, DeLong EF, Herndl GJ, Stepanauskas R (2011) Potential for chemolithoautotrophy among ubiquitous bacteria lineages in the dark ocean. Science, 333, 1296-1300.
Varela MM, van Aken HM, Sintes E, Reinthaler T, Herndl GJ (2011) Contribution of Crenachaeota and Bacteria to autotrophy in the North Atlantic interior. Environmental Microbiology, 13(6), 1524-1533.
4.1. Observation of dense shelf, deep, and bottom waters