Dark Carbon Fixation in the Columbia River's Estuarine Turbidity Maxima: Molecular Characterization of Red-Type cbbL Genes and Measurement of DIC Uptake Rates in Response to Added Electron Donors

Dark CO₂ fixation has been shown to rival the importance of oxygenic photosynthesis in the global carbon cycle, especially in stratified environments, such as salt wedge estuaries. We investigated this process in the Columbia River estuary using a variety of techniques including functional gene cloning of cbbL (the large subunit of form I RuBisCO), quantitative real-time PCR (qPCR) estimations of cbbL abundance, and analyses of stimulated ¹⁴C-bicarbonate assimilation. A diversity of red-type cbbL genes were retrieved from clone libraries, with 28 unique operational taxonomic units determined from 60 sequences. The majority of the sequences formed two clusters that were distinct from the major clusters typically found in soil environments, revealing the presence of a unique community of autotrophic or facultatively autotrophic/mixotrophic microorganisms in the Columbia River estuary. qPCR estimates indicated that roughly 0.03-0.15 % of the microbial population harbored the cbbL gene, with greater numbers of total bacteria and cbbL gene copies found in the estuarine turbidity maxima (ETM) compared to non-ETM events. In vitro incubations with radiolabeled bicarbonate indicated maximum stimulation by thiosulfate and also suggested that a diversity of other potential electron donors may stimulate CO₂ fixation, including nitrite, ammonium, and Mn(II). Taken together, these results highlight the diversity of the microbial metabolic strategies employed and emphasize the importance of dark CO₂ fixation in the dynamic waters of the Columbia River estuary despite the abundance of organic material.