Feeding the Microbial Loop: the role of manganese oxide minerals in organic matter degradation and bacterial community activity
Proposed research and background
Manganese-oxidizing microbes are ubiquitous in both marine and freshwater environments. The manganese oxide minerals precipitated by their activity are strong sorbents and oxidants, capable of degrading recalcitrant organic matter and altering the speciation of trace metals. How and why microbes oxidize manganese (Mn) and precipitate Mn oxides remains enigmatic, however, as no known organism derives energy from the reaction. My thesis research thus far explores the how component of this question, investigating the role of exuded biological molecules as (1) ligands that stabilize the intermediate Mn(III) species allowing complete oxidation from Mn(II) to Mn(IV) and (2) nucleation sites or templates for mineral growth.
With funding from the Coastal Ocean Institute, I hope to address the why component of microbially catalyzed Mn oxidation. One previous study observed the production of small organic molecules such as pyruvate and formaldehyde following reaction of Mn oxides and seawater (Sunda and Kieber 1994). This result led the authors to hypothesize that microbes catalyze Mn oxidation in order to initiate oxidative degradation of recalcitrant organic molecules into labile molecules available for consumption—effectively using something inorganic to re-introduce carbon from larger molecules, such as biopolymers, into the microbial loop. This hypothesis is difficult to test, however, as it would require demonstrating that the same microbes catalyzing Mn oxidation are also benefitting by getting to consume the substrate produced. As we have not yet identified the diversity of microbes responsible for environmental Mn oxidation, we cannot track the fate of the oxidizers. I propose to test an intermediate hypothesis: that the reaction of Mn oxides and organic matter does degrade recalcitrant organic carbon into small, labile molecules and that these labile molecules are consumed by bacteria, generating a measurable response in the bacterial metatranscriptome.
Experiments testing this hypothesis will be modeled on previous work using incubations to demonstrate rapid shifts in community structure and activity following the addition of novel carbon sources (McCarren et al. 2010, Nelson and Carlson 2012) but will add Mn oxides rather than excess carbon, thereby altering both the quality and quantity of carbon compounds available to organisms. Support from my lab is available to run these experiments and to monitor changes in the composition of organic matter over the course of the experiments. This objective will be accomplished by utilizing FT-ICR-MS and NMR to obtain both molecular and bulk scale characterization of organic matter. My adviser, Colleen Hansel, has an active user award at Environmental Molecular Sciences Laboratory (EMSL) at Pacific Northwest National Laboratory which gives me instrument access over the next two years that will facilitate this portion of the experiment. I am requesting funding to conduct metatranscriptomic analysis before and after reaction of seawater with Mn oxides following the method outlined in McCarren et al. 2010.
Manganese in coastal sediments undergoes 100-300 redox cycles per atom before burial (Canfield et al. 1993), remineralizing organic carbon. In estuarine environments with suboxic or anoxic bottom water, cycling will begin in the water column. Does the microbial community respond to this input of labile carbon? We know that Mn oxides can and will degrade organic matter; my experiments as currently planned will document the extent of organic matter alteration and identify any specificity towards certain classes of molecules.
Having funding from the Coastal Ocean Institute to track how this reaction in turn affects the bacterial community will allow us to comprehend the broader impact of Mn cycling and gage its importance as a process in estuarine environments. This study is especially important in light of increasing coastal zone eutrophication and the need to develop management strategies. I hope to use the findings to estimate the net, equilibrium effect of Mn cycling: remineralization or increased production. Ultimately, I would like to test additional environments (i.e. tropical vs. temperate estuaries) and to compare the microbes benefitting from the addition of degraded carbon to that initiating the oxidation of Mn.
To test my hypothesis, I propose several incubations run in parallel. To start, estuarine water will be collected and filtered to remove microbes. A portion of the water will then be reacted with Mn oxides for 96 hours and then re-filtered to remove Mn oxide particles as the presence of particles could provoke a transcriptomic response. I plan to use three different concentrations of Mn oxides—25, 1, and 0.1 µM. 0.1 µM is comparable or slightly higher than environmental concentrations in estuaries and coastal waters while 25 µM is high enough that I will be able to track the reaction’s progress spectrophotometrically, using two methods I have standardized and currently work with to measure remaining Mn(III/IV) oxides as well as dissolved Mn(II) and Mn(III). Mn oxides will be added as the mineral phase birnessite, which is highly reactive and a common product of bacterial Mn oxidation.
This Mn-reacted seawater will be used as the experimental incubation. A control incubation will use another portion of the filtered seawater and contain a concentration of reduced Mn equivalent to that found in the reacted seawater as a result of oxide reduction. This will control for any microbial response to elevated dissolved metal concentrations. A second control will contain no added Mn. All incubations will then be re-inoculated with unfiltered water and reacted for 48 hours, at which time I will collect samples for carbon characterization and metatranscriptomics. Growth will be monitored via flow cytometry throughout the experiment. Incubations will be run in duplicate, producing 14 samples for transcriptome analysis plus 2 additional ‘time zero’ samples of the raw estuarine water.
Following pricing available on the UC Davis Genome Center website (http://dnatech.genomecenter.ucdavis.edu/prices.html, likely comparable to other facilities), all samples can be analyzed via Illumina Mi-Seq for $1389.
Canfield, D.E., Thamdrup, B. & Hansen, J. The anaerobic degradation of organic matter in Danish coastal sediments: Iron reduction, manganese reduction, and sulfate reduction. Geochimica et Cosmochimica Acta 57, 3867-3883 (1993).
McCarren, J. et al. Microbial community transcriptomes reveal microbes and metabolic pathways associated with dissolved organic matter turnover in the sea. Proceedings of the National Academy of Sciences 107, 16420-16427 (2010).
Nelson, C.E. and Carlson, C.A. Tracking differential incorporation of dissolved organic carbon types among diverse lineages of Sargasso Sea bacterioplankton. Environmental Microbiology 14, 1500-1516 (2012).
Sunda, W. G. & Kieber, D. J. Oxidation of humic substances by manganese oxides yields low-molecular-weight organic substrates. Nature 367, 62-64 (1994).